Cellulase variants

ABSTRACT

Described is a method for improving the properties of a cellulolytic enzyme by amino acid substitution, deletion or insertion, the method comprising the steps of:  
     a. constructing a multiple alignment of at least two amino acid sequences known to have three-dimensional structures similar to endoglucanase V (EGV) from  Humicola insolens  known from Protein Data Bank entry 4ENG;  
     b. constructing a homology-built three-dimensional structure of the cellulolytic enzyme based on the structure of the EGV;  
     c. identifying amino acid residue positions present in a distance from the substrate binding cleft of not more than 5 Å;  
     d. identifying surface-exposed amino acid residues of the enzyme;  
     e. identifying all charged or potentially charged amino acid residue positions of the enzyme;  
     f. choosing one or more positions wherein the amino acid residue is to be substituted, deleted or where an insertion is to be provided; and  
     g. carrying out the substitution, deletion or insertion by using conventional protein engineering techniques. Also described are cellulase variants obtained by this method.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation application of PCT/DK97/00393 filed Sep.17, 1997 and claims priority under 35 U.S.C. 119 of Danish application1013/96 filed Sep. 17, 1996, the contents of which are fullyincorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to cellulase variants, i.e.endo-β-1,4-glucanase variants, derived from a parental cellulase, i.e.endo-β-1,4-glucanase, by substitution, insertion and/or deletion, whichvariant has a catalytic core domain, in which the variant at position 5holds an alanine residue (A), a serine residue (S), or a threonineresidue (T); at position 8 holds a phenylalanine residue (F), or atyrosine residue (Y); at position 9 holds a phenylalanine residue (F), atryptophan residue (W), or a tyrosine residue (Y); at position 10 holdsan aspartic acid residue (D); and at position 121 holds an aspartic acidresidue (D).

BACKGROUND ART

[0003] Cellulases or cellulolytic enzymes are enzymes involved inhydrolyses of cellulose. In the hydrolysis of native cellulose, it isknown that there are three major types of cellulase enzymes involved,namely cellobiohydrolase (1,4-β-D-glucan cellobiohydrolase, EC3.2.1.91), endo-β-1,4-glucanase (endo-1,4-β-D-glucan 4-glucanohydrolase,EC 3.2.1.4) and β-glucosidase (EC 3.2.1.21).

[0004] Especially the endo-β-1,4-glucanases (EC No. 3.2.1.4) constitutean interesting group of hydrolases for the mentioned industrial uses.Endoglucanases catalyses endo hydrolysis of 1,4-β-D-glycosidic linkagesin cellulose, cellulose derivatives (such as carboxy methyl celluloseand hydroxy ethyl cellulose), lichenin, β-1,4 bonds in mixed β-1,3glucans such as cereal β-D-glucans or xyloglucans and other plantmaterial containing cellulosic parts. The authorized name isendo-1,4-β-D-glucan 4-glucano hydrolase, but the abbreviated termendoglucanase is used in the present specification. Reference can bemade to T.- M. Enveri, “Microbial Cellulases” in W. M. Fogarty,Microbial Enzymes and Biotechnology, Applied Sciences Publishers, p.183-224 (1983); Methods in Enzymology, (1988) Vol. 160, p. 200-391(edited by Wood, W. A. and Kellogg, S. T.); Béguin, P., “MolecularBiology of Cellulose Degradation”, Annu. Rev. Microbiol. (1990), Vol.44, pp. 219-248; Béguin, P. and Aubert, J-P., “The biologicaldegradation of cellulose”, FEMS, Microbiology Reviews 13 (1994) p.25-58; Henrissat, B., “Cellulases and their interaction with cellulose”,Cellulose (1994), Vol. 1, pp. 169-196.

[0005] Cellulases are synthesized by a large number of microorganismswhich include fungi, actinomycetes, myxobacteria and true bacteria butalso by plants. Especially endoglucanases of a wide variety ofspecificities have been identified

[0006] A very important industrial use or cellulolytic enzymes is theuse for treatment of cellulosic textile or fabric, e.g. as ingredientsin detergent compositions or fabric softener compositions, forbio-polishing of new fabric (garment finishing), and for obtaining a“stone-washed” look of cellulose-containing fabric, especially denim,and several methods for such treatment have been suggested, e.g. inGB-A-1 368 599, EP-A-0 307 564 and EP-A-0 435 876, WO 91/17243, WO91/10732, WO 91/17244, PCT/DK95/000108 and PCT/DK95/00132. Anotherimportant industrial use of cellulolytic enzymes is the use fortreatment of paper pulp, e.g. for improving the drainage or for deinkingof recycled paper.

[0007] It is also known that cellulases may or may not have a cellulosebinding domain (a CBD). The CBD enhances the binding of the enzyme to acellulose-containing fiber and increases the efficacy of the catalyticactive part of the enzyme

[0008] Fungi and bacteria produces a spectrum of cellulolytic enzymes(cellulases) which, on the basis of sequence similarities (hydrophobiccluster analysis), can be classified into different families of glycosylhydrolases [Henrissat B & Bairoch A; Biochem. J. 1993 293 781-788]. Atpresent are known cellulases belonging to the families 5, 6, 7, 8, 9,10, 12, 26, 44, 45, 48, 60, and 61 of glycosyl hydrolases.

[0009] Industrially well-performing endo-β-1,4-glucanases are describedin e.g. WO 91/17243, WO 91/17244 and WO 91/10732, and specific cellulasevariants are described in WO 94/07998.

[0010] It is an object of the present invention to provide novelvariants of cellulolytic enzymes, which variants, when compared to theparental enzyme, show improved performance.

SUMMARY OF THE INVENTION

[0011] In a cellulolytic enzyme useful in industrial processes, i.e. anendo-β-1,4-glucanase, a number of amino acid residue positions importantfor the properties of the enzyme and thereby for the performance thereofin these processes has been identified.

[0012] Accordingly, in a first aspect the present invention provides amethod for improving the properties of a cellulolytic enzyme by aminoacid substitution, deletion or insertion, the method comprising thesteps of:

[0013] a. constructing a multiple alignment of at least two amino acidsequences known to have three-dimensional structures similar toendoglucanase V (EGV) from Humicola insolens known from Protein DataBank entry 4ENG;

[0014] b. constructing a homology-built three-dimensional structure ofthe cellulolytic enzyme based on the structure of the EGV;

[0015] c. identifying amino acid residue positions present in a distancefrom the substrate binding cleft of not more than 5 Å;

[0016] d. identifying surface-exposed amino acid residues of the enzyme;

[0017] e. identifying all charged or potentially charged amino acidresidue positions of the enzyme;

[0018] f. choosing one or more positions wherein the amino acid residueis to be substituted, deleted or where an insertion is to be provided;

[0019] g. carrying out the substitution, deletion or insertion by usingconventional protein engineering techniques.

[0020] By using the method of the invention, it is now possibleeffectively to transfer desirable properties from one cellulase toanother by protein engineering methods which are known per se.

[0021] More particular the invention provides cellulase variantsimproved with respect to altered (increased or decreased) catalyticactivity; and/or altered sensitivity to anionic tensides; and/or alteredpH optimum and pH profile activity-wise as well as stability-wise.

[0022] Accordingly, in a further aspect, the invention provides acellulase variant derived from a parental cellulase by substitution,insertion and/or deletion, which variant has a catalytic core domain, inwhich the variant

[0023] at position 5 holds an alanine residue (A), a serine residue (S),or a threonine residue (T);

[0024] at position 8 holds a phenylalanine residue (F), or a tyrosineresidue (Y);

[0025] at position 9 holds a phenylalanine residue (F), a tryptophanresidue (W), or a tyrosine residue (Y);

[0026] at position 10 holds an aspartic acid residue (D); and

[0027] at position 121 holds an aspartic acid residue (D) (cellulasenumbering).

DETAILED DISCLOSURE OF THE INVENTION

[0028] Cellulase Variants

[0029] The present invention provides new cellulase variants derivedfrom a parental cellulase by substitution, insertion and/or deletion. Acellulase variant of this invention is a cellulase variant or mutatedcellulase, having an amino acid sequence not found in nature. Thecellulase variants of the invention show improved performance, inparticular with respect to increased catalytic activity; and/or alteredsensitivity to anionic tensides; and/or altered pH optimum; and/oraltered thermostability.

[0030] Formally the cellulase variant or mutated cellulase of thisinvention may be regarded a functional derivative of a parentalcellulase (i.e. the native or wild-type enzyme), and may be obtained byalteration of a DNA nucleotide sequence of the parental gene or itsderivatives, encoding the parental enzyme. The cellulase variant ormutated cellulase may be expressed and produced when the DNA nucleotidesequence encoding the cellulase variant is inserted into a suitablevector in a suitable host organism. The host organism is not necessarilyidentical to the organism from which the parental gene originated.

[0031] In the literature, enzyme variants have also been referred to asmutants or muteins.

[0032] Amino Acids

[0033] In the context of this invention the following symbols andabbreviations for amino acids and amino acid residues are used: A = Ala= Alanine C = Cys = Cysteine D = Asp = Aspartic acid E = Glu = Glutamicacid F = Phe = Phenylalanine G = Gly = Glycine H = His = Histidine I =Ile = Isoleucine K = Lys = Lysine L = Leu = Leucine M = Met = MethionineN = Asn = Asparagine P = Pro = Proline Q = Gln = Glutamine R = Arg =Arginine S = Ser = Serine T = Thr = Threonine V = Val = Valine W = Trp =Tryptophan Y = Tyr = Tyrosine B = Asx = Asp or Asn Z = Glx = Glu or GinX = Xaa = Any amino acid * = Deletion or absent amino acid

[0034] Cellulase Numbering

[0035] In the context of this invention a specific numbering of aminoacid residue positions in cellulolytic enzymes is employed. By aligningthe amino acid sequences of known cellulases, as in Table 1 below, it ispossible to unambiguously allot an amino acid position number to anyamino acid residue in any cellulolytic enzyme, if its amino acidsequence is known.

[0036] In Table 1, below, 11 selected amino acid sequences of cellulasesof different microbial origin are aligned. These are (a) Humicolainsolens; (b) Acremonium sp.; (c) Volutella collectotrichoides; (d)Sordaria fimicola; (e) Thielavia terrestris; (f) Fusarium oxysporum; (g)Myceliophthora thermophila; (h) Crinipellis scabella; (i) Macrophominaphaseolina; (j) Pseudomonas fluorescens; (k) Ustilago maydis. Thecellulases (a-i) are described in WO 96/29397, (j) is described inGeneBank under the accession number G45498, and (k) is described inGeneBank under the accession number S81598 and in Biol. Chem.Hoppe-Seyler 1995 376 (10) 617-625.

[0037] Using the numbering system originating from the amino acidsequence of the cellulase (endo-β-l,4-glucanase) obtained from thestrain of Humicola insolens DSM 1L800, disclosed in e.g. WO 91/17243,which sequence is shown in the first column of Table 1, aligned with theamino acid sequence of a number of other cellulases, it is possible toindicate the position of an amino acid residue in a cellulolytic enzymeunambiguously.

[0038] In describing the various cellulase variants produced orcontemplated according to the invention, the following nomenclatures areadapted for ease of reference:

[0039] [original amino acid; Position; Substituted amino acid]

[0040] Accordingly, the substitution of glutamine with histidine inposition 119 is designated as Q119H.

[0041] Amino acid residues which represent insertions in relation to theamino acid sequence of the cellulase from Humicola insolens, arenumbered by the addition of letters in alphabetical order to thepreceding cellulase number, such as e.g. position *21aV for the“inserted” valine (V), where no amino acid residue is present, betweenlysine at position 21 and alanine at position 22 of the amino acidsequence of the cellulase from Humicola insolens, cf. Table 1.

[0042] Deletion of a proline (P) at position 49 in the amino acidsequence of the cellulase from Humicola insolens is indicated as P49*.

[0043] Multiple mutations are separated by slash marks (″/″), e.g.Q119H/Q146R, representing mutations in positions 119 and 146substituting glutamine (Q) with histidine (H), and glutamine (Q) witharginine (R), respectively.

[0044] If a substitution is made by mutation in e.g. a cellulose derivedfrom a strain of Humicola insolens, the product is designed e.g.“Humicola insolens/*49P”.

[0045] All positions referred to in this application by cellulosenumbering refer to the cellulase numbers described above, and aredetermined relative to the amino acid sequence of the cellulase derivedfrom Humicola insolens, cf. Table 1, (a). TABLE 1 Amino Acid SequenceAlignment; Cellulase Numbering of Selected Cellulases of DifferentMicrobial Origin (a) Humicola insolens; (b) Acremonium sp.; (c)Volutella collectotorichoides; (d) Sordaria fimicola; (e) Thielaviaterrestris; (f) Fusarium oxysporum; (g) Myceliphthora thermophila; (h)Crinipellis scabella; (i) Macrophomina phaseolina; (j) Pseudomonasfluorescens; (k) Ustilago maydis.      a b c d e f g h i j k  1    A G GG G G G T T C * 2    D S T S S S I A S N * 3    G G G G G G G G G G G4    R H R K Q H Q V V Y M 5    S T T S S S T T T A A 6    T T T T T T TT T T T 7    R R R R R R R R R R R 8    Y Y Y Y Y Y Y Y Y Y Y 9    W W WW W W W W W W W 10   D D D D D D D D D D D 11   C C C C C C C C C C C12   C C C C C C C C C C C 13   K K K K K K K K K K L 14   P P P P P P PP P P A 15   S S S S S S S S S H S 16   C C C C C C C C C C A 17   G A GA A S A G A G S 18   W W W W W W W W W W W 19   A D D S P S P S T S E20   K E E G G G G G G A G 21   K K K K K K K K K N K21a  * * * * * * * * * V * 22   A A A A A A G A A P A 23   P A S S A A PS S S P 24   V V V V V V * V V L V 25   N S S N S N S S S V Y 26   Q R QR Q A S A K S A 27   P P P P P P P P P P P 28   V V V V V A V V V L V29   F T K L Y L Q R G Q D 30   S T T A A T A T T S A 31   C C C C C C CC C C C 32   N D D D D D D D D S K 33   A R R A A K K R I A A 34   N N NN N N N N N N D 35   F N N N F D D G D N G 36   Q S N N Q N N N N T V37   R P P P R P P T A R T 38   I L L L L I F L Q L L 39   T S A N S S NG T S I 40   D P S D D N D P P D D 41   F * * A F T G * S V S 42   D G TN N N G * D S K 42a  * * * * * * S D L * K 43   A A A V V A T V L * K44   K V R K Q V R K K G P 45   S S S S S N S S S S S 46   G G G G G G GG S S G 47   C C C C C C C C C C Q 48   E D D D N E D D D D S 49   P PS * * G A S * * G 49a  * * * * * * * * * * C 49b  * * * * * * * * * * N50   G N N G G G G G G G G 51   G G G G G G G G G G G 52   V V V S S S ST S G N 53   A A A A A A A S A G K 54   Y F Y Y Y Y Y F Y Y F 55   S T TT S A M T Y M M 56   C C C C C C C C C C C 57   A N N A A T S A S W S58   D D D N D N S N N D C 59   Q N N N Q Y Q N Q K M 60   T Q Q W T S SG G I Q 61   P P P P P P P P P P P 62   W W W W W W W F W F F 63   A A AA A A A A A A D 64   V V V V V V V I V V D 65   N N N N N N S D N S E66   D N D D D D D N D P T 67   D N N N N E E N S T D 68   F V L L L L LT L L P 69   A A A A A A S A S A T 70   L Y Y Y Y Y Y Y Y Y L 71   G G GG G G G G G G A 72   F F F F F F W F F Y F 73   A A A A A A A A A A G74   A A A A A A A A A A F 75   T T T T T T V A A T G 76   S A A K S K KH K S A 77   I F F L I I L L L S F 78   A P S S A S A A S G T 79   G G GG G G G G G D T 80   S G G G G G S S K V G 81   N N S T S S S S Q * Q82   E E E E E E E E E * E 83   A A A S S A S A T * S 84   G S S S S S QA D * D 85   W W W W W W W W W * T 86   C C C C C C C C C C D 87   C C CC C C C C C G C 88   A A A A A A A Q G R A 89   C C C C C C C C C C C90   Y Y Y Y Y Y Y Y Y Y F 91   E A A A A A E E K Q Y 92   L L L L L L LL L L A 93   T Q Q T T T T T T Q E 94   F F F F F F F F F F F 95   T T TT T T T T T T E 95a  * * * * * * * * * G * 95b  * * * * * * * * * S *95c  * * * * * * * * * S * 95d  * * * * * * * * * Y *95e  * * * * * * * * * N * 95f  * * * * * * * * * A *95g  * * * * * * * * * P * 95h  * * * * * * * * * G H95i  * * * * * * * * * D D 95j  * * * * * * * * * P A95k  * * * * * * * * * G Q 96   S S S S S S T S S S G 97   G G G G G G GG T A K 98   P P P P P P P P A A A 99   V V V V V V V V V L M 100  A A AS A K A V S A K 101  G G G G G G G G G G R 102  K K K K K K K K K K N103  K T T T T K K K Q T K 104  M M M L M M M L M M L 105  V V V V V I IT I I I 106  V V V V V V V V V V F 107  Q Q Q Q Q Q Q Q Q Q Q 108  S S SS S S A V I A V 109  T T T T T T T T T T T 110  S N N S S N N N N N N111  T T T T T T T T T I V 112  G G G G G G G G G G G 113  G G G G G G GG G Y G 114  D D D D D D D D D D D 115  L L L L L L L L L V V 116  G S SG G G G G G S Q 117  S G G S S D D N N G S 118  N T N N N N N N N G Q119  H H H H Q H H H H Q H 120  F F F F F F F F F F F 121  D D D D D D DD D D D 122  L I I L I L L L I I F 123  N Q L N A M A M A L O 124  I M MM M M I I M V I 125  P P P P P P P P P P P 126  G G G G G G G G G G G127  G G G G G G G G G G G 128  G G G G G G G G G G G 129  V L L V V V VV V V L 130  G G G G G G G G G G G 131  I I I L I I I L I A A 132  F F FF F F F F F F F 132a * * * * * * * T * * P 133  D D D D N D N Q N N K134  G G G G G G A G G A G 135  C C C C C C C C C C C 136  T T T K S T TP S S P 137  P P P R S S D A K A A 138  Q Q Q E Q E Q Q Q Q Q 139  F F WF F F Y F W W W 140  G G G G G G G G N G G 140a * F V * * K A S G V V141  G T S G G A P W I S E 142  L F F L L L P N * N A 143  P P P P P G NG * A S 143a * * * * * * G * N E L 143b * * * * * * W * L L W 144  G G GG G G G G G G G 145  Q N N A A A D A N A D 146  R R R Q Q Q R Q Q Q Q147  Y Y Y Y Y Y Y Y Y Y Y 148  G G G G G G G G G G G 149  G G G G G G GG G G G 150  I T T I I I V F F F V 150a * * * * * * * * * L *150b * * * * * * * * * A * 150c * * * * * * * * * A *150d * * * * * * * * * C * 150e * * * * * * * * * K *150f * * * * * * * * * Q * 150g * * * * * * * * * Q *150h * * * * * * * * * L * 150i * * * * * * * * * G *150j * * * * * * * * * Y * 150k * * * * * * * * * N * 151  S T T S S S HS T A K 152  S S S S S S S S D S S 153  R R R R R R K R R L A 154  N S SS D S E D S S T 155  E Q Q E Q E E Q Q Q E 156  C C C C C C C C C Y C157  D A S D D D E S A K S 158  R E Q S S S S Q S T K 159  F L I F F Y FL L C L 160  P P P P P P P P P V P 160a * * * * * * * * * L *160b * * * * * * * * * N * 160c * * * * * * * * * R *160d * * * * * * * * * C * 160e * * * * * * * * * D *160f * * * * * * * * * S * 160g * * * * * * * * * V *160h * * * * * * * * * F * 160i * * * * * * * * * G *160j * * * * * * * * * S * 160k * * * * * * * * * R *160l * * * * * * * * * G * 160m * * * * * * * * * L * 161  D S S A A E EA S T K 162  A V A A P L A A K Q P 163  L L L L L L L V W L L 164  K R QK K K K Q Q Q Q 165  P D P P P D P A A Q E 166  G G G G G G G G S G G167  C C C C C C C C C C C 168  Y H N Q Q H N Q N T K 169  W W W W W W WF W W W 170  R R R R R R R R R F R 171  F Y Y F F F F F P A F 172  D D DD D D D D D E S 173  W W W W W W W W W W E 174  F F F F F F F M F F W175  K N N K Q E Q C E E G 176  N D D N N N N G N A D 177  A A A A A A AA A A N 178  D D D D D D D D U D P 179  N N N N N N N N N N V 180  P P PP P P P P P P L 181  S N D E T D S N T S K 182  F V V F F F V V V L G183  S N S T T T T T D K S 184  F W W F F F F F W Y P 185  R R R K Q E QR E K K 186  Q R R Q Q Q E P P E R 187  V V V V V V V V V V V 188  Q R QQ Q Q A T T P K 189  C C C C C C C C C C C 190  P P P P P P P P P P P191  A A A S A K S A Q A K 192  E A A E E A S Q B E S 193  L L L L I L LI L L L 194  V T T T V L T T V T I 195  A N D S A D S N A T D 196  R R RR R I K I R R R 197  T S T T S S S S T S S 198  G C G G C G G C C G C199  C C C C C C C C C M C 200  R V R K K K S V S N Q

[0046] The Enzyme (Endo-β-1,4-Glucanase) Variants of the Invention

[0047] The present invention relates to cellulase variants. Morespecifically the present invention provides cellulase variant derivedfrom a parental cellulase by substitution, insertion and/or deletion,which variant has a catalytic core domain, in which the variant

[0048] at position 5 holds an alanine residue (A), a serine residue (S),or a threonine residue (T);

[0049] at position 8 holds a phenylalanine residue (F), or a tyrosineresidue (Y);

[0050] at position 9 holds a phenylalanine residue (F), a tryptophanresidue (W), or a tyrosine residue (Y);

[0051] at position 10 holds an aspartic acid residue (D); and

[0052] at position 121 holds an aspartic acid residue (D) (cellulasenumbering).

[0053] The endoglucanase of the invention may comprise a cellulosebinding domain (CBD) existing as an integral part of the enzyme, or aCBD from another origin may be introduced into the endoglucanase thuscreating an enzyme hybride. In this context, the term “cellulose-bindingdomain” is intended to be understood as defined by Peter Tomme et al.“Cellulose-Binding Domains: Classification and Properties” in “EnzymaticDegradation of Insoluble Carbohydrates”, John N. Saddler and Michael H.Penner (Eds.), ACS Symposium Series, No. 618, 1996. This definitionclassifies more than 120 cellulose-binding domains into 10 families(I-X), and demonstrates that CBDs are found in various enzymes such ascellulases, xylanases, mannanases, arabinofuranosidases, acetyl(esterases and chitinases. CBDs have also been found in algae, e.g. thered alga Porphyra purpurea as a non-hydrolytic polysaccharide-bindingprotein, see Tomme et al., op.cit. However, most of the CBDs are fromcellulases and xylanases, CBDs are found at the N and C termini ofproteins or are internal. Enzyme hybrids are known in the art, see e.g.WO 90/00609 and WO 95/16782, and may be prepared by transforming into ahost cell a DNA construct comprising at least a fragment of DNA encodingthe cellulose-binding domain ligated, with or without a linker, to a DNAsequence encoding the endoglucanase and growing the host cell to expressthe fused gene.

[0054] Enzyme hybrids may be described by the following formula:

CBD-MR-X or X-MR-CBD

[0055] wherein CBD is the N-terminal or the C-terminal region of anamino acid sequence corresponding to at least the cellulose-bindingdomain; MR is the middle region (the linker), and may be a bond, or ashort linking group preferably of from about 2 to about 100 carbonatoms, more preferably of from 2 to 40 carbon atoms; or is preferablyfrom about 2 to about 100 amino acids, more preferably of from 2 to 40amino acids; and X is an N-terminal or C-terminal region of the enzymeaccording to the invention.

[0056] The Method of the Invention

[0057] In another aspect, the present invention relates to a method forimproving the properties of a cellulolytic enzyme by amino acidsubstitution, deletion or insertion, the method comprising the steps of:

[0058] a. constructing a multiple alignment of at least two amino acidsequences known to have three-dimensional structures similar toendoglucanase V (EGV) from Humicola insolens known from Protein DataBank entry 4ENG;

[0059] b. constructing a homology-built three-dimensional structure ofthe cellulolytic enzyme based on the structure of the EGV;

[0060] c. identifying amino acid residue positions present in a distancefrom the substrate binding cleft of not more than 5 Å;

[0061] d. identifying surface-exposed amino acid residues of the enzyme;

[0062] e. identifying all charged or potentially charged amino acidresidue positions of the enzyme;

[0063] f. choosing one or more positions wherein the amino acid residueis to be substituted, deleted or where an insertion is to be provided;

[0064] g. carrying out the substitution, deletion or insertion by usingconventional protein engineering techniques.

[0065] Step f. of the method is preferably carried out by choosingpositions which, as a result of the alignment of step a., carry the sameamino acid residue in a majority of the aligned sequences; morepreferably in at least 63% of the aligned sequences; even morepreferably positions which, in the aligned sequences, carries differentamino acid residues, cf. below.

[0066] In a preferred embodiment, the specific activity of the cellulasecan be improved, preferably by carrying out a substition, deletion orinsertion at amino acid residue positions present in a distance from thesubstrate binding cleft of not more than 5 Å, more preferably not morethan 3 Å, even more preferably not more than 2.5 Å. It is believed thatresidues present in a distance of not more than 2.5 Å are capable ofbeing in direct contact with the substrate.

[0067] In another preferred embodiment, the pH activity profile, the pHactivity optimum, the pH stability profile, or the pH stability optimumof the cellulase can be altered, preferably by carrying out asubstitution, deletion or insertion at amino acid residue positionspresent either in a distance from the substrate binding cleft of notmore than 5 Å, more preferably not more than 3 Å, even more preferablynot more than 2.5 Å; or at surface-exposed amino acid residue positionsof the enzyme, thereby altering the electrostatic environment eitherlocally or globally. It is preferred to perform a substitution involvinga charged or potentially charged residue, this residue either being theoriginal residue or the replacement residue. In the present context,charged or potentially charged residues are meant to include: Arg, Lys,His, Cys (if not part of a disulfide bridge), Tyr, Glu, Asp.

[0068] In yet another preferred embodiment, the stability of thecellulase in the presence of an anionic tenside or anionic detergentcomponent can be altered, preferably by carrying out a substitution,deletion or insertion at surface-exposed amino acid residue positions ofthe enzyme, thereby altering the electrostatic environment eitherlocally or globally. It is preferred to perform a substitution involvinga charged or potentially charged residue, this residue either being theoriginal residue or the replacement residue. In the present context,charged or potentially charged residues are meant to include: Arg, Lys,His, Cys (if not part of a disulfide bridge), Tyr, Glu, Asp. Mutationstowards a more negatively charged aa residue result in improvedstability of the cellulase in the presence of an anionic tenside,whereas mutations towards a more positively charged aa residue decreasesthe stability of the cellulase towards anionic tensides.

[0069] Further, cellulase variants comprising any combination of two ormore of the amino acid substitutions, deletions or insertions disclosedherein are also within the scope of the present invention, cf. theexemplified variants.

[0070] Multiple Sequence Alignment of Cellulases

[0071] The multiple sequence alignment is performed using the Pileupalgorithm as implemented in the Wisconsin Sequence Analysis Packageversion 8.1-UNIX (GCG, Genetics Computer Group, Inc.). The method usedis similar to the method described by Higgens and Sharp (CARBIOS 1989 5151-153). A gap creation penalty of 3.0 and a gap extension penalty of0.1 is used together with a scoring matrix as described in Nucl. AcidsRes. 1986 14 (16) 6745-6763 (Dayhoff table (Schwartz, R. M. and Dayhoff,M. O.; Atlas of Protein Sequence and Structure (Dayhoff, M. O. Ed.);National Biomedical Research Foundation, Washington D.C. 1979 353-358)rescaled by dividing each value by the sum of its row and column, andnormalizing to a mean of 0 and standard deviation of 1.0. The value forFY (Phe-Tyr)=1.425. Perfect matches are set to 1.5 and no matches on anyrow are better than perfect matches).

[0072] Pair-Wise Sequence Alignment of Cellulases

[0073] A pair-wise sequence alignment is performed using the algorithmdescribed by Needleman & Wunsch (J. Mol. Biol. 1970 48 443-453), asimplemented in the GAP routine in the Wisconsin Sequence AnalysisPackage (GCG). The parameters used for the GAP routine are the same asmentioned for the Pileup routine earlier.

[0074] Pair-Wise Sequence Alignment of Cellulases with Forced Pairing

[0075] A pair-wise sequence alignment with forced pairing of residues isperformed using the algorithm described by Needleman & Wunsch (J. Mol.Biol. 1970 48 443-453), as implemented in the GAP routine in theWisconsin Sequence Analysis Package (GCG). The parameters used for theGAP routine are the same as mentioned for the Pileup routine earlier,where the scoring matrix is modified to incorporate a residue named Xwhich symbolize the residues to be paired. The diagonal value for Xpaired with X is set to 9.0 and all off diagonal values involving X isset to 0.

[0076] Complex between Humicola insolens Endoglucanase and Celloheptaose

[0077] Based on the X-ray structure of the core domain of the Humicolainsolens EGV endoglucanase inactive variant (D10N) in complex withcellohexaose (Davies et. al.; Biochemistry 1995 34 16210-12220, PDBentry 4ENG) a model of the structure of the native Humicola insolens EGVendoglucanase core domain in complex with celloheptaose is build usingthe following steps:

[0078] 1. Using the Biopolymer module of the Insight II 95.0 (Insight II95.0 User Guide, October 1995. San Diego: Biosym/MSI, 1995) replace N10with a aspartic acid.

[0079] 2. Make a copy of the sugar unit occupying subsite −3 by copyingall the molecule and delete the extra atoms. Manually move the new sugarunit to best fit the unoccupied −1 binding site. Create the bonds tobind the new sugar unit to the two existing cellotriose units.

[0080] 3. Delete overlapping crystal water molecules. These areidentified by using the Subset Interface By_Atom 2.5 command.

[0081] 4. Build hydrogens at a pH of 8.0 and applying charged terminals

[0082] 5. Protonate D121 using the Residue Replace <D121 residuename>ASP L command.

[0083] 6. Apply the CVFF forcefield template through the commandPotentials Fix.

[0084] 7. Fix all atoms except the new sugar unit.

[0085] 8. Relax the atomic position of the new sugar unit using 300cycles of simple energy minimization followed by 5000 steps of 1 fssimple molecular dynamics ending by 300 cycles of simple energyminimization all using the molecular mechanics program Discover95.0/3.0.1 (Discover 95.0/3.0.0 User Guide, October 1995. San Diego:Biosym/MSI, 1995.).

[0086] Homology Building of Cellulases

[0087] The construction of a structural model of a cellulase with knownamino acid sequence based on a known X-ray structure of the Humicolainsolens EGV cellulase consists of the following steps:

[0088] 1. Define the approximate extend of the core region of thestructure to be modeled and the alignment of the cysteine based onmultiple sequence alignment between many known industrially usefulcellulase sequences.

[0089] 2. Pair-wise sequence alignment between the new sequence and thesequence of the known X-ray structure.

[0090] 3. Define Structurally Conserved Regions (SCRs) based on thesequence alignment.

[0091] 4. Assign coordinates for the model structure within the SCRs.

[0092] 5. Find structures for the loops or Variable Regions (VRs)between the SCRs by a search in a loop structure database.

[0093] 6. Assign coordinates for the VRs in the model structure from thedatabase search result.

[0094] 7. Create disulfide bonds and set protonation state.

[0095] 8. Refine the build structure using molecular mechanics.

[0096] The known X-ray structure of the Humicola insolens EGV cellulasewill in the following be termed the reference structure. The structureto be modeled will be termed the model structure.

[0097] Ad 1: The approximate extent of the core part of the enzyme isdetermined by a multiple sequence alignment including many knowncellulase sequences. Since the reference structure only contain atomiccoordinates for the core part of the enzyme only the residues in thesequence to be modeled which align with the core part of the referencestructure can be included in the model building. This alignment alsodetermines the alignment of the cysteine. The multiple sequencealignment is performed using the Pileup algorithm as described earlier.

[0098] Ad 2: A pair-wise sequence alignment is performed as describedearlier. If the cysteine in the conserved disulfide bridges and/or theactive site residues (D10 and D121) does not align, a pair-wise sequencealignment using forced pairing of the cysteines in the conserveddisulfide bridges and/or the active site residues is performed asdescribed earlier. The main purpose of the sequence alignment is todefine SCRs (see later) to be used for a model structure generation.

[0099] Ad 3: Based on the sequence alignment Structurally ConservedRegions (SCRs) are defined as continuous regions of overlapping sequencewith no insertions or deletions.

[0100] Ad 4: Using the computer program Homology 95.0 (Homology UserGuide, October 1995. San Diego: Biosym/MSI, 1995.) atomic coordinates inthe model structure can be generated from the atomic coordinates of thereference structure using the command AssignCoords Sequences.

[0101] Ad 5: Using the computer program Homology 95.0 possibleconformations for the remaining regions, named Variable Regions (VRs)are found by a search in the loop structure database included inHomology 95.0. This procedure is performed for each VR.

[0102] Ad 6: If the VR length is smaller than six residues the firstloop structure in the database search result is selected for coordinategeneration. In cases where longer loops are generated the first solutionin the list which does not have severe atomic overlap are selected. Thedegree of atomic overlap can be analyzed using the Bump Monitor AddIntra command in the computer program Insight II 95.0 (Insight II 95.0User Guide, October 1995. San Diego: Biosym/MSI, 1995.) a parameter of0.25 for the Bump command will show the severe overlap. If more than tenbumps exists between the inserted loop region and the remaining part ofthe protein the next solution is tested. If no solution is found withthese parameters, the solution with the fewest bumps is selected. Thecoordinates for the VR regions are generated using the commandAssignCoords Loops in the program Homology 95.0.

[0103] Ad 7: The disulfide bonds are created using the Bond Createcommand in the Biopolymer module of Insight II 95.0 and the protonationstate is set to match pH 8.0 with charged caps using the Hydrogenscommand. Finally the active proton donor (the residue equivalent to D121in the reference structure) is protonated using the residue replace<D121 residue name> ASP L command. To finalize the data of the model theappropriate forcefield template is applied using the CVFF forcefieldthrough the command Potentials Fix.

[0104] Ad 8: Finally the modeled structure is subjected to 500 cyclesenergy minimization using the molecular mechanics program Discover95.0/3.0.1 (Discover 95.0/3.0.0 User Guide, October 1995. San Diego:Biosym/MSI, 1995.). The output from the above described procedure isatomic coordinates describing a structural model for the core domain ofa new cellulase based on sequence homology to the Humicola insolens EGVcellulase.

[0105] Superpositioning of Cellulase Structures

[0106] To overlay two cellulase structures a superposition of thestructures are performed using the Structure Alignment command of theHomology 95.0 (Homology User Guide, October 1995. San Diego: Biosym/MSI,1995.). All parameters for the command are chosen as the default values.

[0107] Determination of Residues within 3 Å and 5 Å from the Substrate

[0108] In order to determine the amino acid residues within a specifieddistance from the substrate, a given cellulase structure is superimposedon the cellulase part of the model structure of the complex betweenHumicola insolens EGV endoglucanase and celloheptaose as describedabove. The residues within a specified distance of the substrate arethen found using the Interface Subset command of the Insight II 95.0(Insight II 95.0 User Guide, October 1995. San Diego: Biosym/MSI, 1995).The specified distance are supplied as parameter to the program.

[0109] The results of this determination are presented in Tables 2 and 3below.

[0110] Determination of Surface Accessibility

[0111] To determine the solvent accessibility the Access_Surf command inHomology 95.0 (Homology User Guide, October 1995; San Diego: Biosym/MSI,1995) was used. The program uses the definition proposed by Lee andRichards (Lee, B. & Richards, F. M. “The interpretation of proteinstructures: Estimation of static accessibility”, J. Mol. Biol. 1971 55379-400). A solvent probe radius of 1.4 Å was used and only heavy atoms(i.e. non-hydrogen atoms) were included in the calculation. Residueswith zero accessibility is defined as being buried, all other residuesare defined as being solvent exposed and on the surface of the enzymestructure.

[0112] Transferring Level of Specific Activity between Cellulases

[0113] In order to transfer the level of catalytic activity between twocellulases, the following protocol is applied using the methodsdescribed above. This method will pinpoint amino acid residuesresponsible for the difference in specific activity, and one or more ofthose amino acid residues must be replaced in one sequence in order totransfer the level of specific activity from the comparison cellulase:

[0114] 1) Perform multiple sequence alignment of all known industiallyuseful cellulases (excluding the Trichoderma reesei cellulases). Fromthis identify conserved disulfide bridges amongst the two involvedsequences and the sequence of the Humicola insolens EGV cellulase areidentified and the active site residues (D10 and D121) are located;

[0115] 2) Perform pair-wise sequence alignment of each sequence with theHumicola insolens EGV cellulase core domain (residues 1-201). If thecysteines in the conserved disulfide bridges does not align at the samepositions and/or if the two active site residues (D10 and D121) does notalign at the same positions then use the pair-wise sequence alignment ofcellulases with forced pairing method. Include only residues in thesequences overlapping with the core domain (residues 1-201) of theHumicola insolens EGV cellulase;

[0116] 3) Create a homology build structure of each sequence;

[0117] 4) Determination of residues within 3 Å from the substrate ineach of the homology build structures. Differences between the sequencesin these positions will most probably be the residues responsible forthe difference in specific activity. In the case where residues ininserts are found in any of the sequences within the above mentioneddistance, the complete insert can be responsible for the difference inspecific activity, and the complete insert must be transferred to thesequence without the insert or the complete insert must be deleted inthe sequence with the insert;

[0118] 5) If not all specific activity was restored by substitution ofresidues within 3 Å of the substrate, determination of residues within 5Å from the substrate in each of the homology build structures willreveal the most probable residues responsible for the remainingdifference in specific activity. In the case where residues in insertsare found in any of the sequences within the above mentioned distance,the complete insert can be responsible for the difference in specificactivity, and the complete insert must be transferred to the sequencewithout the insert or the complete insert must be deleted in thesequence with the insert.

[0119] Transferring the Level of Stability towards Anionic Tensidesbetween Cellulases

[0120] In order to transfer level of stability towards anionic tensidesbetween two cellulases, the following protocol is applied using themethods described above. This method will pinpoint amino acid residuesresponsible for the difference in level of stability towards anionictensides, and one or more of those amino acid residues must be replacedin one sequence in order to transfer the level of specific activity fromthe comparison cellulase:

[0121] 1) Perform multiple sequence alignment of all known industriallyuseful cellulases (excluding Trichoderma reesei cellulases). From thisidentify conserved disulfide bridges amongst the two involved sequencesand the sequence of the Humicola insolens EGV cellulase are identifiedand the active site residues (D10 and D121) are located;

[0122] 2) Perform pair-wise sequence alignment of each sequence with theHumicola insolens EGV cellulase core domain (residues 1-201). If thecysteines in the conserved disulfide bridges does not align at the samepositions and/or if the two active site residues (D10 and D121) does notalign at the same positions then use the pair-wise sequence alignment ofcellulases with forced pairing method. Include only residues in thesequences overlapping with the core domain (residues 1-201) of theHumicola insolens EGV cellulase;

[0123] 3) Create a homology build structure of each sequence;

[0124] 4) Determination of residues located at the surface of theenzyme. This is done by calculation the surface accessibility. Residueswith a surface accessibility greater than 0.0 Å² are exposed to thesurface;

[0125] 5) Any residue exposed to the surface belonging to the followinggroup of amino acids: D, E, H, K, R and C if not involved in disulfidebridge which differ between the two sequences will most probably beresponsible for the difference in level of stability towards anionictensides. In the case where residues in inserts are found in any of thesequences within the above mentioned group of amino acid types, thecomplete insert can be responsible for the difference in level ofstability towards anionic tensides, and the complete insert must betransferred to the sequence without the insert or the complete insertmust be deleted in the sequence with the insert.

[0126] Disulfide Bridges

[0127] Disulfide bridges (i.e. Cys-Cys bridges) stabilize the structureof the enzyme. It is believed that a certain number of stabilizingdisulfide bridges are necessary to maintain the a proper stability ofthe enzyme. However, it is also contemplated that disulfide bridges canbe removed from the protein structure resulting in an enzyme variantwhich is less stable, especially less thermostable, but which still hassignificant activity.

[0128] Therefore, in another aspect, the invention provides a cellulasevariant which variant holds 4 or more of the following disulfidebridges: C11-C135; C12-C47; C16-C86; C31-C56; C87-C199; C89-C189; andC156-C167 (cellulase numbering). In a more specific embodiment thevariant of the invention holds 5 or more of the following disulfidebridges: C11-C135; C12-C47; C16-C86; C31-C56; C87-C199; C89-C189; andC156-C167 (cellulase numbering). In its most specific embodiment, thevariant of the invention holds 6 or more of the following disulfidebridges: C11-C135; C12-C47; C16-β-C86; C31-C56; C87-C199; C89-C189; andC156-C167 (cellulase numbering).

[0129] In another embodiment the invention provides a cellulase variantin which cysteine has been replaced by another natural amino acid at oneor more of the positions 16, 86, 87, 89, 189, and/or 199 (cellulasenumbering).

[0130] Binding Cleft Substitutions

[0131] In a further aspect, the invention provides a cellulase variantderived from a parental cellulase by substitution, insertion and/ordeletion at one or more amino acid residues located in the substratebinding cleft. Mutations introduced at positions close to the substrateaffect the enzyme-substrate interactive bindings.

[0132] An appropriate way of determining the residues interacting with apotential substrate in a structure is to partitionate the structure in“shells”. The shells are defined as: 1st shell are residues directlyinteracting with the substrate, i.e. closest inter atomic distancebetween substrate and residue both including hydrogen atoms are smallerthan 2.5 Å which will include all direct interaction via hydrogen bondsand other non bonded interactions. The subsequent (2nd, 3rd e.t.c.)shells are defined in the same way, as the residues with inter atomicdistances smaller than 2.5 Å to the substrate or all previouslydetermined shells. In this way the structure will be partitioned inshells. The routine “subset zone” in the program Insight II 95.0(Insight II 95.0 User Guide, October 1995. San Diego: Biosym/MSI, 1995.)can be used to determine the shells.

[0133] In a preferred embodiment, the amino acid residue contemplatedaccording to this invention is located in the substrate binding cleft ata distance of up to 5 Å from the substrate.

[0134] When subjecting the aligned cellulases to the computer modelingmethod disclosed above, the following positions within a distance of upto 5 Å from the substrate are revealed: 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 18, 19, 20, 21, 21a, 42, 44, 45, 47, 48, 49, 49a, 49b,74, 82, 95j, 110, 111, 112, 113, 114, 115, 116, 119, 121, 123, 127, 128,129, 130, 131, 132, 132a, 133, 145, 146, 147, 148, 149, 150b, 178,and/or 179 (cellulase numbering), cf. Table 2.

[0135] Accordingly, in a more specific embodiment, the inventionprovides a cellulase variant which has been derived from a parentalcellulase by substitution, insertion and/or deletion at one or more ofthese acid residues. In a particular 25 embodiment, the cellulasevariant is derived from one of the cellulases identified in Table 2 ((a)Humicola insolens; (b) Acremonium sp.; (c) Volutella collectotrichoides;(d) Sordaria fimicola; (e) Thielavia terrestris; (f) Fusarium oxysporum;(g) Myceliophthora thermophila; (h) Crinipellis scabella; (i)Macrophomina phaseolina; (j) Pseudomonas fluorescens; (k) Ustilagomaydis), by substitution, insertion and/or deletion at one or more ofthe positions identified in Table 2 for these cellulases. TABLE 2 AminoAcid Residues less than 5 Å from the Substrate Positions Identified byCellulase Numbering (a) Humicola maclens; (b) Acremonium ap.; (c)Volutella collectotrichoides; (d) Sordaria fimicola; (e) Thielaviaterrestris; (f) Fusarium oxysporum; (g) Myceliophthora thermophila; (h)Crinipellis scabella; (I) Macrophomina phaseolina; (j) Pseudomoriasfluorescens; (k) Ustilago maydis.      a b c d e f g h i j k 4                      Y 5    S T T S S S T T T A A 6    T T T T T T T TT T T 7    R R R R R R R R R R R 8    Y Y Y Y Y Y Y Y Y Y Y 9    W W W WW W W W W W W 10   D D D D D D D D D D D 11               C 12   C C C CC C C C C C C 13   K K K K K K K K K K L 14   P P P P P P P P P P A15   S S S S S S S S S H S 16                     C 18   W W W W W W W WW W W 19   A D D S P S P S   T E 20   K E E   G   G G   A 21   K K K K KK K K K   K 21a                    V 42     G T           D 44     V R KQ V R     G 45   S S S S S N S S S S 47   C C C C C C C C C C 48   E D DD N E D D D D S 49     P 49a                      C49b                      N 74   A A A A A A A A A A F 82   E E E E EE   E E   E 95j                    P 110  S N N S S N N N N N N 111  T TT T T T T T T I V 112  G G G G G G G G G G G 113  G G G G G G G G G Y G114  D D D D D D D D D D D 115  L L L L L L L L L V V 116          G119  H H H H Q H H H H Q N 121  D D D D D D D D D D D 123    Q 127  G GG G G G G G G G G 128  G G G G C G C C G C G 129  V L L V V V V V V V L130  G C C G G G G G C C G 131  I I I L I I I L I A A 132  F F F F F F FF F F F 132a               T     P 133                  N N145                    A D 146  R R R Q Q Q R Q Q Q Q 147  Y Y Y Y Y Y YY Y Y Y 148  G G G G G G G G G G G 149  G     G G G     G150b                   A 178  D D D D D D D D D D P 179  N N N N N N N NN N V

[0136] In another preferred embodiment, the amino acid residuecontemplated according to this invention is located in the substratebinding cleft at a distance of up to 3 Å from the substrate.

[0137] When subjecting the aligned cellulases to the computer modelingmethod disclosed above, the following positions within a distance of upto 3 A from the substrate are revealed: 6, 7, 8, 10, 12, 13, 14, 15, 18,20, 21, 45, 48, 74, 110, 111, 112, 113, 114, 115, 119, 121, 127, 128,129, 130, 131, 132, 132a, 147, 148, 150b, 178, and/or 179 (cellulasenumbering). cf. Table 3.

[0138] Accordingly, in a more specific embodiment, the inventionprovides a cellulase variant which has been derived from a parentalcellulase by substitution, insertion and/or deletion at one or more ofthese acid residues. In a particular embodiment, the cellulase variantis derived from one of the cellulases identified in Table 3 ((a)Humicola insolens; (b) Acremonium sp.; (c) Volutella collectotrichoides;(d) Sordaria fimicola; (e) Thielavia terrestris; (f) Fusarium oxysporum;(g) Myceliophthora thermophila; (h) Crinipellis scabella; (i)Macrophomina phaseolina; (j) Pseudomonas fluorescens; (k) Ustilagomaydis), by substitution, insertion and/or deletion at one or more ofthe positions identified in Table 3 for these cellulases. TABLE 3 AminoAcid Residues less than 3 Å from the Substrate Positions Identif led byCellulase Numbering (a) Humi cola maclens; (b) Acremonium sp.; (c)Volutella collectotrichoides; (d) Sordaria fimicola; (e) Thielaviaterrestris; (f) Fusarium oxysporum; (g) Myceliophthora thermophila; (h)Crinipellis scabella; (I) Macrophomina phaseolina; (j) Pseudomonasfluorescens; (k) Ustilago maydis.      a b c d e f g h i j k  6    T T TT T T T T T T T 7    R R R R R R R R R R R 8    Y Y Y Y Y Y Y Y Y Y Y10   D D D D D D D D   D D 12   C C C C C C C C C C C 13   K K KK   K     K   L 14     P     P P P P P   A 15   S S S S S S S S S H S18   W W W W W W W W W W W 20     E E 21   K     K 45   S S S S S N S SS S S 48     D     N E D     D 74   A     A A A A A A   F 110    N N   SN N N N N N 111  T T T T T T T T T 112  G G G G G G G G G G G 113  G G GG       G G Y G 114  D D D D D D D D D D D 115  L L L L L L L L L V V119  H H H   Q H   H   Q 121  D D D D D D D D D D D 127  G     G G GG   G 128  G     G G G G G G   G 129  V L L V V V V V V V L 130  G G G GG G G G G G G 131  I I I L I I I L I A A 132  F F F F F F F F F F F132a               T     P 146          Q     Q   Q Q 147  Y Y Y Y Y Y YY Y Y Y 148  G G G G G G G G G G G 150b                   A 178  D D DD       D   D P 179  N N N N N N N N N N

[0139] Partly Conserved Amino Acid Residues

[0140] As defined herein a “partly conserved amino acid residue” is anamino acid residue identified according to Table 1, at a position atwhich position between 7 to 10 amino acid residues of the 11 residues(i.e. more than 63%) indicated in Table 1 for that position, areidentical.

[0141] Accordingly, the invention further provides a cellulase variant,in which variant an amino acid residue has been changed into a conservedamino acid residue at one or more positions according to Table 1, atwhich position(s) between 7 and 10 amino acid residues of the 11residues identified in Table 1, are identical.

[0142] In a preferred embodiment the invention provides a cellulasevariant, which has been derived from a parental cellulase bysubstitution, insertion and/or deletion at one or more of the followingpositions: 13, 14, 15, 20, 21, 22, 24, 28, 32, 34, 45, 48, 50, 53, 54,62, 63, 64, 65, 6E6, 68, 69, 70, 71, 72, 73, 74, 75, 79, 85, 88, 90, 92,93, 95, 96, 97, 98, 99, 104, 106, 110, 111, 113, 115, 116, 118, 119,131, 134, 138, 140, 146, 152, 153, 163, 166, 169, 170, 171, 172, 173,174, 174, 177, 178, 179, 180, 193, 196, and/or 197 (cellulasenumbering).

[0143] In a more specific embodiment the invention provides a cellulasevariant that has been subjected to substitutions, insertions and/ordeletions, so as to comprise one or more of the amino acid residues atthe positions identified in Table 4, below. The positions in Table 4reflects the “partly conserved amino acid residue positions” as well asthe non-conserved positions present within 5 Å of the substrate inbinding cleft all of which indeed are present in the aligned sequencesin Table 1. TABLE 4 Selected Substitutions, Insertions and/or Dele-tions Positions Identified by Cellulase Numbering Position Amino AcidResidue   4 R, H, K, Q, V, Y, M   5 S, T, A  13 K, L  14 P, A  15 H, S 16 C, A  19 A, D, S, P, T, E  20 A, E, G, K  21 K, N  21a V, *  22 A,G, P  24 *, L, V  28 A, L, V  32 D, K, N, S  34 D, N  38 F, I, L, Q  42D, G, T, N, S, K, *  44 K, V, R, Q, G, P  45 N, S  46 G, S  47 C, Q  48D, E, N, S  49 P, S, A, G, *  49a C, *  49b N, *  50 G, N  53 A, G, K, S 54 F, Y  62 F, W  63 A, D  64 D, I V  65 D, E, N, S  66 D, N, P, T  68F, L, P, T, V  69 A, S, T  70 L, Y  71 A, G  72 F, W, Y  73 A, G  74 A,F  75 A, G, T, V  79 G, T  82 E, *  88 A, G, Q, R  90 F, Y  92 A, L  93E, Q, T  95 E, T  95j P, *  96 S, T  97 A, G, T  98 A, P  99 L, V 104 L,M 106 F, V 110 N, S 111 I, T, V 113 G, Y 115 L, V 116 G, Q, S 118 G, N,Q, T 119 H, N, Q 129 L, V 131 A, I, L 132 A, P, T, * 133 D, K, N, Q 134A, G 138 E, Q 145 A, D, N, Q 146 Q, R 150b A, * 152 D, S 153 A, K, L, R163 L, V, W 166 G, S 169 F, W 170 F, R 171 A, F, Y 172 D, E, S 173 E, W174 F, M, W 177 A, N 178 D, P 179 N, V 180 L, P 193 T, L 196 T, K, R 197S, T

[0144] In a yet more preferred embodiment, the invention provides acellulase variant derived from a parental cellulase by substitution,insertion and/or deletion at one or more amino acid residues asindicated in Tables 5-6, below. The cellulase variant may be derivedfrom any parental cellulase holding the amino acid residue stated at theposition indicated. In particular the parental cellulase may be aHumicola insolens cellulase; an Acremonium sp. Cellulase; a Volutellacollectotrichoides cellulase; a Sordaria fimicola cellulase; a Thielaviaterrestris cellulase; a Fusariuim oxysporum cellulase; a Myceliophthorathermophila cellulase; a Crinipellis scabella cellulase; a Macrophominaphaseolina cellulase; a Pseudomonas fluorescens cellulase; or a Ustilagomaydis cellulase.

[0145] Moreover, the cellulase variant may be characterized by havingimproved performance, in particular with respect to

[0146] 1. improved performance defined as increased catalytic activity;

[0147] 2. altered sensitivity to anionic tenside; and/or

[0148] 3. altered pH optimum;

[0149] as also indicated in Tables 5-6. The positions listed in Table 5reflect transfer of properties between the different cellulases alignedin Table 1. The positions listed in Table 6 reflect transfer ofproperties from Humicola insolens EGV to the other cellulases aligned inTable 1. TABLE 5 Preferred Cellulase Variants Positions Identified byCellulase Numbering K13L, L13K (1, 2, 3); P14A, A14P (1); S15H, H15S (1,3); K20E, K20G, K20A, E20K, G20K, A20K, E20G, E20A, G20E, A20E, G20A,A20G (1, 2, 3); K21N, N21K (1, 2, 3); A22G, A22P, G22A, P22A, G22P, P22G(1); V24*, V24L, *24V, L24V, *24L, L24* (1); V28A, V28L, A28V, L28V,A28L, L28A (1); N32D, N32S, N32K, D32N, S32N, K32N, D32S, D32K, S32D,K32D, S32K, K32S (2, 3); N34D, D34N (2); I38L, I38F, I38Q, L38I, F38I,Q38I, L38F, L38Q, F38L, Q38L, F38Q, Q38F (1) S45N, N45S (1); G46S, S46G(1); E48D, E48N, D48E, N48E, D48N, N48D (1, 2, 3); G50N, N50G (1); A53S,A53G, A53K, S53A, G53A, K53A, S53G, S53K, G53S, K53S, G53K, K53G (1);Y54F, F54Y (1, 3); W62F, F62W (1, 2); A63D, D63A (2, 3); V64I, V64D,I64V, D64V, I64D, D64I (2); N65S, N65D, N65E, S65N, D65N, E65N, S65D,S65E, D65S, E65S, D65E, E65D (2); D66N, D66P, D66T, N66D, P66D, T66D,N66P, N66T, P66N, T66N, P66T, T66P (2, 3); F68V, F68L, F68T, F68P, V68F,L68F, T68F, P68F, V68L, V68T, V68P, L68V, T68V, P68V, L68T, L68P, T68L,P68L, T68P, P68T (1, 2); A69S, A69T, S69A, T69A, S69T, T69S (1); L70Y,Y70L (1); G71A, A71G (1); F72W, F72Y, W72F, Y72F, W72Y, Y72W (1); A73G,G73A (1); A74F, F74A (1); T75V, T75A, T75G, V75T, A75T, G75T, V75A,V75G, A75V, G75V, A75G, G75A (1); G79T, T79G (1); W85T, T85W (1); A88Q,A88G, A88R, Q88A, G88A, R88A, Q88G, Q88R, G88Q, R88Q, G88R, R88G (1, 2,3); Y90F, F90Y (1); L92A, A92L (1); T93Q, T93E, Q93T, E93T, Q93E, E93Q(2); T95E, E95T (2); S96T, T96S (1); G97T, G97A, T97G, A97G, T97A, A97T(1); P98A, A98P (1); V99L, L99V (1); M104L, L104M (1); V106F, F106V (1,3); S110N, N110S (1); T111I, T111V, I111T, V111T, I111V, V111I (1);G113Y, Y113G (1, 3); L115V, V115L (1); G116S, G116Q, S116G, Q116G,S116Q, Q116S (1); N118T, N118G, N118Q, T118N, G118N, Q118N, T118G,T118Q, G118T, Q118T, G118Q, Q118G (1); H119Q, H119N, Q119H, N119H (1,2); V129L, L129V (1); I131L, I131A, L131I, A131I, L131A, A131L (1);G134A, A134G (1); Q138E, E138Q (1, 2, 3); G140N, N140G (1); R146Q, Q146R(1, 2, 3); S152D, D152S (2); R153K, R153L, R153A, K153R, L153R, A153R,K153L, K153A, L153K, A153K, L153A, A153L (2); L163V, L163W, V163L,W163L, V163W, W163V (1); G166S, S166G (1); W169F, F169W (1); R170F,F170R (1, 2, 3); F171Y, F171A, Y171F, A171F, Y171A, A171Y (1); D172E,D172S, E172D, S172D, E172S, S172E (2); W173E, E173W (1, 2, 3); F174M,F174W, M174F, W174F, M174W, W174M (1); A177N, N177A (1); D178P, P178D(1, 2, 3); N179V, V179N (1); P180L, L180P (1); L193I, I193L (1); R196I,R196K, I196R, K196R, I196K, K196I (2, 3); T197S, S197T (1)

[0150] TABLE 6 Preferred Cellulase Variants Positions Identified byCellulase Numbering L13K (1, 2, 3); A14P (1); H15S (1, 3); E20K, G20K,A20K (1, 2, 3); N21K (1, 2, 3); G22A, P22A (1); *24V, L24V (1); A28V,L28V (1); D32N, S32N, K32N (2, 3); D34N (2); L38I, F38I, Q38I (1); N45S(1); S46G (1); D48E, N48E (1, 2, 3); N50G (1); S53A, G53A, K53A (1);F54Y (1, 3); F62W (1, 2); D63A (2, 3); I64V, DG4V (2); S65N, D65N, E65N(2) N66D, R66D, T66D (2, 3); V68F, L68F, T68F, P68F (1, 2); S69A, T69A(1) Y70L (1) A71G (1) W72F, Y72F (1) G73A (1) F74A (1) V75T, A75T, G75T(1) T79G (1); T85W (1); Q88A, G88A, R88A (1, 2, 3) F90Y (1) A92L (1)Q93T, E93T (2); E95T (2); T96S (1); T97G, A97G (1); A98P (1); L99V (1);L104M (1); F106V (1, 3); N110S (1); I111T, V111T (1); Y113G (1, 3);V115L (1); S116G, Q116G (1); T118N, G118N, Q118N (1); Q119H, N119H (1,2); L129V (1); L131I, A131I (1); A134G (1); E138Q (1, 2, 3); N140G (1);Q146R (1, 2, 3); D152S (2); K153R, L153R, A153R (2); V163L, W163L (1);S166G (1); F169W (1); P170R (1, 2, 3); Y171F, A171F (1); E172D, S172D(2); E173W (1, 2, 3); M174F, W174F (1); N177A (1); P178D (1, 2, 3);V179N (1); L180P (1); I193L (1); I196R, K196R (2,3); S197T (1)

[0151] Altered Sensibility Towards Anionic Tensides

[0152] As mentioned above, anionic tensides are products frequentlyincorporated into detergent compositions. Sometimes cellulolytic enzymeshaving an increased stability towards anionic tensides is a desire, andsometimes cellulolytic enzymes having an increased sensitivity arepreferred. In a further aspect the invention provides cellulase variantsof an altered anionic tenside sensitivity.

[0153] Accordingly, a cellulase variant of the invention of alteredanionic tenside sensitivity is a cellulase variant which has beenderived from a parental cellulase by substitution, insertion and/ordeletion at one or more of the following positions: 2, 4, 7, 8, 10, 13,15, 19, 20, 21, 25, 26, 29, 32, 33, 34, 35, 37, 40, 42, 42a, 43, 44, 48,53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 70, 72, 76, 79, 80, 82, 84, 86,88, 90, 91, 93, 95, 95d, 95h, 95j, 97, 100, 101, 102, 103, 113, 114,117, 119, 121, 133, 136, 137, 138, 139, 140a, 141, 143a, 145, 146, 147,150e, 150j, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160c, 160e,160k, 161, 162, 164, 165, 168, 170, 171, 172, 173, 175, 176, 178, 181,183, 184, 185, 186, 188, 191, 192, 195, 196, 200, and/or 201 (cellulasenumbering). These positions contain, in at least one of the cellulasesequences aligned in Table 1, a charged or potentially charged aaresidue.

[0154] In a particular embodiment, the cellulase variant is derived fromone of the cellulases identified in Table 7, below, ((a) Humicolainsolens; (b) Acremonium sp.; (c) Volutella collectotrichoides; (d)Sordaria fimicola; (e) Thielavia terrestris; (f) Fusarium oxysporum; (g)Myceliophthora thermophila; (h) Crinipellis scabella; (i) Macrophominaphaseolina; (j) Pseudomonas fluorescens; (k) Ustilago maydis), bysubstitution, insertion and/or deletion at one or more of the positionsidentified in Table 7 for these cellulases. TABLE 7 Altered Sensitivitytowards Anionic Tensides Positions identified by Cellulase Numbering (a)Humicola irisolens; (b) Acremonium sp.; (c) Volutellacollectotrichoides; (d) Sordaria fimicola; (e) Thielavia terrestris; (f)Fusarium oxysporum; (g) Myceliophthora thermophila; (h) Cririipellisscabella; (i) Macrophomina phaseolina; (j) Pseudomonas fluorescens; (k)Ustilago maydis.      a b c d e f g h i j k  2    D 4    R H RK   H       Y   7    R R R R R R R R R R R 8    Y Y Y Y Y Y Y Y Y Y Y10   D D D D D D D D D D D 13   K K K K K K K K K K K15                     H 19     D D               E 20   K E E 21   K KK K K K K K K   K 25                       Y 26     R   R         K29       K   Y     R     D 32     D D D D D D D D   K 33     R R     K KR 34                       D 35             D D   D37   R       R         R 40   D     D D   D     D D42  D               D   K 42a                 D     K43                       D 44   K   R K     R K K 48   E D D D   E D D DD 53                       K 54   Y   Y Y Y Y Y   Y Y55                   Y 58   D D D   D         D 59             Y       K63                       D 64                       D65                 D     E 66   D   D D D D D   D 67   D         EE       D 70     Y Y Y Y Y Y Y Y Y 72                     Y76         K   K K H K 79                     D 80                   K82   E E E E E E E E E   E 84                   D   D86                       D 88                     R 90   Y Y Y Y Y Y Y YY Y 91   E           E E K   Y 93                       E95                       E 95d                    Y95h                      H 95i                    D D97                       K 100            K         K101                      R 102  K K K K K K K K K K 103  K         K KK     K 113                    Y 114  D D D D D D D D D D D117            D D 119  H H H H   H H H H 121  D D D D D D D D D D D133  D D D D D   D       K 136        K 137        R     D   K138        E   E 139                Y 140a           K141                      E 143a                   E L145             D        D 146  R R R       R 147  Y Y Y Y Y Y Y Y Y Y Y150e                   K 150j                   Y151              H       K 152                  D 153  R R R R R K R R154          D   E D 155  E     E   E E       E 156                    Y157  D     D D D E     K 158  R E                 K 159            Y160c                   R 160e                   D160k                   R 161  D         E E       K162                  K 164  K R   K K K K 165    D       D         E168  Y H       H         K 170  R R R R R R R R R   R 171    Y Y 172  DD D D D D D D D E 173                      E 175  K     K   E     E E176    D D               D 178  D D D D D D D D D D 181      DE   D         K 183                  D K 184                    Y 185  RR R K   E   R E K K 186    R R       E     E R188    R                 K 191            K         K 192  E     EE   E   E E 195      D     D         D 196  R R R R R   K   R R R200  R   R K K K 201  R R R R R R R R R R R

[0155] In a still further aspect, the present invention relates to anenzyme composition comprising an enzyme exhibiting cellulolytic activityas described above.

[0156] The enzyme composition of the invention may, in addition to thecellulase of the invention, comprise one or more other enzyme types, forinstance hemi-cellulase such as xylanase and mannanase, other cellulasecomponents, chitinase, lipase, esterase, pectinase, cutinase, phytase,oxidoreductase, peroxidase, laccase, oxidase, pactinmethylesterase,polygalacturonase, protease, or amylase.

[0157] The enzyme composition may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the enzyme composition may be in the form ofa granulate or a microgranulate. The enzyme to be included in thecomposition may be stabilized in accordance with methods known in theart.

[0158] Examples are given below of preferred uses of the enzymecomposition of the invention. The dosage of the enzyme composition ofthe invention and other conditions under which the composition is usedmay be determined on the basis of methods known in the art.

[0159] The enzyme composition according to the invention may be usefulfor at least one of the following purposes.

[0160] Uses

[0161] During washing and wearing, dyestuff from dyed fabrics or garmentwill conventionally bleed from the fabric which then looks faded andworn. Removal of surface fibers from the fabric will partly restore theoriginal colors and looks of the fabric. By the term “colorclarification”, as used herein, is meant the partly restoration of theinitial colors of fabric or garment throughout multiple washing cycles.

[0162] The term “de-pilling” denotes removing of pills from the fabricsurface.

[0163] The term “soaking liquor” denotes an aqueous liquor in whichlaundry may be immersed prior to being subjected to a conventionalwashing process. The soaking liquor may contain one or more ingredientsconventionally used in a washing or laundering process.

[0164] The term “washing liquor” denotes an aqueous liquor in whichlaundry is subjected to a washing process, i.e. usually a combinedchemical and mechanical action either manually or in a washing machine.Conventionally, the washing liquor is an aqueous solution of a powder orliquid detergent composition.

[0165] The term “rinsing liquor” denotes an aqueous liquor in whichlaundry is immersed and treated, conventionally immediately after beingsubjected to a washing process, in order to rinse the laundry, i.e.essentially remove the detergent solution from the laundry. The rinsingliquor may contain a fabric conditioning or softening composition.

[0166] The laundry subjected to the method of the present invention maybe conventional washable laundry. Preferably, the major part of thelaundry is sewn or un-sewn fabrics, including knits, wovens, denims,yarns, and toweling, made from cotton, cotton blends or natural ormanmade cellulosics (e.g. originating from xylan-containing cellulosefibers such as from wood pulp) or blends thereof. Examples of blends areblends of cotton or rayon/viscose with one or more companion materialsuch as wool, synthetic fibers (e.g. polyamide fibers, acrylic fibers,polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers,polyvinylidene chloride fibers, polyurethane fibers, polyurea fibers,aramid fibers), and cellulose-containing fibers (e.g. rayon/viscose,ramie, flax/linen, jute, cellulose acetate fibers, lyocell).

DETERGENT DISCLOSURE AND EXAMPLES

[0167] Surfactant System

[0168] The detergent compositions according to the present inventioncomprise a surfactant system, wherein the surfactant can be selectedfrom nonionic and/or anionic and/or cationic and/or ampholytic and/orzwitterionic and/or semi-polar surfactants.

[0169] The surfactant is typically present at a level from 0.1% to 60%by weight.

[0170] The surfactant is preferably formulated to be compatible withenzyme components present in the composition. In liquid or gelcompositions the surfactant is most preferably formulated in such a waythat it promotes, or at least does not degrade, the stability of anyenzyme in these compositions.

[0171] Preferred systems to be used according to the present inventioncomprise as a surfactant one or more of the nonionic and/or anionicsurfactants described herein.

[0172] Polyethylene, polypropylene, and polybutylene oxide condensatesof alkyl phenols are suitable for use as the nonionic surfactant of thesurfactant systems of the present invention, with the polyethylene oxidecondensates being preferred. These compounds include the condensationproducts of alkyl phenols having an alkyl group containing from about 6to about 14 carbon atoms, preferably from about 8 to about 14 carbonatoms, in either a straight chain or branched-chain configuration withthe alkylene oxide. In a preferred embodiment, the ethylene oxide ispresent in an amount: equal to from about 2 to about 25 moles, morepreferably from about 3 to about 15 moles, of ethylene oxide per mole ofalkyl phenol. Commercially available nonionic surfactants of this typeinclude IgepalTM CO-630, marketed by the GAF Corporation; and TritonTMX-45, X-114, X-100 and X-102, all marketed by the Rohm & Haas Company.These surfactants are commonly referred to as alkylphenol alkoxylates(e.g., alkyl phenol ethoxylat(es).

[0173] The condensation products of primary and secondary aliphaticalcohols with about 1 to about 25 moles of ethylene oxide are suitablefor use as the nonionic surfactant of the nonionic surfactant systems ofthe present invention. The alkyl chain of the aliphatic alcohol caneither be straight or branched, primary or secondary, and generallycontains from about 8 to about 22 carbon atoms. Preferred are thecondensation products of alcohols having an alkyl group containing fromabout 8 to about 20 carbon atoms, more preferably from about 10 to about18 carbon atoms, with from about 2 to about 10 moles of ethylene oxideper mole of alcohol. About 2 to about 7 moles of ethylene oxide and mostpreferably from 2 to 5 moles of ethylene oxide per mole of alcohol arepresent in said condensation products. Examples of commerciallyavailable nonionic surfactants of this type include TergitolTM 15-S-9(The condensation product of C11-C15 linear alcohol with 9 molesethylene oxide), TergitolTM 24-L-6 NMW (the condensation product ofC12-C14 primary alcohol with 6 moles ethylene oxide with a narrowmolecular weight distribution), both marketed by Union CarbideCorporation; NeodolTM 45-9 (the condensation product of C14-C15 linearalcohol with 9 moles of ethylene oxide), NeodolTM 23-3 (the condensationproduct of C12-C13 linear alcohol with 3.0 moles of ethylene oxide),NeodolTM 45-7 (the condensation product of C14-C15 linear alcohol with 7moles of ethylene oxide), NeodolTM 45-5 (the condensation product ofC14-C15 linear alcohol with 5 moles of ethylene oxide) marketed by ShellChemical Company, KyroTM EOB (the condensation product of C13-C15alcohol with 9 moles ethylene oxide), marketed by The Procter & GambleCompany, and Genapol LA 050 (the condensation product of C12-C14 alcoholwith 5 moles of ethylene oxide) marketed by Hoechst. Preferred range ofHILB in these products is from 8-11 and most preferred from 8-10.

[0174] Also useful as the nonionic surfactant of the surfactant systemsof the present invention are alkylpolysaccharides disclosed in U.S. Pat.No. 4,565,647, having a hydrophobic group containing from about 6 toabout 30 carbon atoms, preferably from about 10 to about 16 carbon atomsand a polysaccharide, e.g. a polyglycoside, hydrophilic group containingfrom about 1.3 to about 10, preferably from about 1.3 to about 3, mostpreferably from about 1.3 to about 2.7 saccharide units. Any reducingsaccharide containing 5 or 6 carbon atoms can be used, e.g., glucose,galactose and galactosyl moieties can be substituted for the glucosylmoieties (optionally the hydrophobic group is attached at the 2-, 3-,4-, etc. positions thus giving a glucose or galactose as opposed to aglucoside or galactoside). The intersaccharide bonds can be, e.g.,between the one position of the additional saccharide units and the 2-,3-, 4-, and/or 6- positions on the preceding saccharide units.

[0175] The preferred alkylpolyglycosides have the formula

R2O (CnH2nO)t(glycosyl)x

[0176] wherein R2 is selected from the group consisting of alkyl,alkylphenyl, hydroxyalkyl, hydroxyalkylphenyl, and mixtures thereof inwhich the alkyl groups contain from about 10 to about 18, preferablyfrom about 12 to about 14, carbon atoms; n is 2 or 3, preferably 2; t isfrom 0 to about 10, preferably 0; and x is from about 1.3 to about 10,preferably from about 1.3 to about 3, most preferably from about 1.3 toabout 2.7. The glycosyl is preferably derived from glucose. To preparethese compounds, the alcohol or alkylpolyethoxy alcohol is formed firstand then reacted with glucose, or a source of glucose, to form theglucoside (attachment at the 1-position). The additional glycosyl unitscan then be attached between their 1-position and the preceding glycosylunits 2-, 3-, 4-, and/or 6-position, preferably predominantly the2-position.

[0177] The condensation products of ethylene oxide with a hydrophobicbase formed by the condensation of propylene oxide with propylene glycolare also suitable for use as the additional nonionic surfactant systemsof the present invention. The hydrophobic portion of these compoundswill preferably have a molecular weight from about 1500 to about 1800and will exhibit water insolubility. The addition of polyoxyethylenemoieties to this hydrophobic portion tends to increase the watersolubility of the molecule as a whole, and the liquid character of theproduct is retained up to the point where the polyoxyethylene content isabout 50% of the total weight of the condensation product, whichcorresponds to condensation with up to about 40 moles of ethylene oxide.Examples of compounds of this type include certain of the commerciallyavailable PluronicTM surfactants, marketed by BASF.

[0178] Also suitable for use as the nonionic surfactant of the nonionicsurfactant system of the present invention, are the condensationproducts of ethylene oxide with the product resulting from the reactionof propylene oxide and ethylenediamine. The hydrophobic moiety of theseproducts consists of the reaction product of ethylenediamine and excesspropylene oxide, and generally has a molecular weight of from about 2500to about 3000. This hydrophobic moiety is condensed with ethylene oxideto the extent that the condensation product contains from about 40% toabout 80% by weight of polyoxyethylene and has a molecular weight offrom about 5,000 to about 11,000. Examples of this type of nonionicsurfactant include certain of the commercially available TetronicTMcompounds, marketed by BASF.

[0179] Preferred for use as the nonionic surfactant of the surfactantsystems of the present invention are polyethylene oxide condensates ofalkyl phenols, condensation products of primary and secondary aliphaticalcohols with from about 1 to about 25 moles of ethyleneoxide,alkylpolysaccharides, and mixtures hereof. Most preferred are C8-C14alkyl phenol ethoxylates having from 3 to 15 ethoxy groups and C8-C18alcohol ethoxylates (preferably C10 avg.) having from 2 to 10 ethoxygroups, and mixtures thereof.

[0180] Highly preferred nonionic surfactants are polyhydroxy fatty acidamide surfactants of the formula

[0181] wherein R1 is H, or R1 is C1-4 hydrocarbyl, 2-hydroxyethyl,2-hydroxypropyl or a mixture thereof, R2 is C5-31 hydrocarbyl, and Z isa polyhydroxyhydrocarbyl having a linear hydrocarbyl chain with at least3 hydroxyls directly connected to the chain, or an alkoxylatedderivative thereof. Preferably, R1 is methyl, R2 is straight C11-15alkyl or C16-18 alkyl or alkenyl chain such as coconut alkyl or mixturesthereof, and Z is derived from a reducing sugar such as glucose,fructose, maltose or lactose, in a reductive amination reaction.

[0182] Highly preferred anionic surfactants include alkyl alkoxylatedsulfate surfactants. Examples hereof are water soluble salts or acids ofthe formula RO(A)mSO3M wherein R is an unsubstituted C10-C-24 alkyl orhydroxyalkyl group having a C10-C24 alkyl component, preferably aC12-C20 alkyl or hydroxyalkyl, more preferably C12-C18 alkyl orhydroxyalkyl, A is an ethoxy or propoxy unit, m is greater than zero,typically between about 0.5 and about 6, more preferably between about0.5 and about 3, and M is H or a cation which can be, for example, ametal cation (e.g., sodium, potassium, lithium, calcium, magnesium,etc.), ammonium or substituted-ammonium cation. Alkyl ethoxylatedsulfates as well as alkyl propoxylated sulfates are contemplated herein.Specific examples of substituted ammonium cations include methyl-,dimethyl, trimethyl-ammonium cations and quaternary ammonium cationssuch as tetramethyl-ammonium and dimethyl piperdinium cations and thosederived from alkylamines; such as ethylamine, diethylamine,triethylamine, mixtures thereof, and the like. Exemplary surfactants areC12-C18 alkyl polyethoxylate (1.0) sulfate (C12-C18E(1.0)M), C12-C18alkyl polyethoxylate (2.25) sulfate (C12-C18(2.25)M, and C12-C18 alkylpolyethoxylate (3.0) sulfate (C12-C18E(3.0)M), and C12-C18 alkylpolyethoxylate (4.0) sulfate (C12-C18E(4.0)M), wherein M is convenientlyselected from sodium and potassium.

[0183] Suitable anionic surfactants to be used are alkyl ester sulfonatesurfactants including linear esters of C8-C20 carboxylic acids (i.e.,fatty acids) which are sulfonated with gaseous SO3 according to “TheJournal of the American Oil Chemists Society”, 52 (1975), pp. 323-329.Suitable starting materials would include natural fatty substances asderived from tallow, palm oil, etc.

[0184] The preferred alkyl ester sulfonate surfactant, especially forlaundry applications, comprise alkyl ester sulfonate surfactants of thestructural formula:

[0185] wherein R3 is a C8-C20 hydrocarbyl, preferably an alkyl, orcombination thereof, R4 is a C1-C6 hydrocarbyl, preferably an alkyl, orcombination thereof, and M is a cation which forms a water soluble saltwith the alkyl ester sulfonate. Suitable salt-forming cations includemetals such as sodium, potassium, and lithium, and substituted orunsubstituted ammonium cations, such as monoethanolamine,diethonolamine, and triethanolamine. Preferably, R3 is C10-C16 alkyl,and R4 is methyl, ethyl or isopropyl. Especially preferred are themethyl ester sulfonates wherein R3 is C10-C16 alkyl.

[0186] Other suitable anionic surfactants include the alkyl sulfatesurfactants which are water soluble salts or acids of the formula ROSO3Mwherein R preferably is a C10-C24 hydrocarbyl, preferably an alkyl orhydroxyalkyl having a C10-C20 alkyl component, more preferably a C12-C18alkyl or hydroxyalkyl, and M is H or a cation, e.g., an alkali metalcation (e.g. sodium, potassium, lithium), or ammonium or substitutedammonium (e.g. methyl-, dimethyl-, and trimethyl ammonium cations andquaternary ammonium cations such as tetramethyl-ammonium and dimethylpiperdinium cations and quaternary ammonium cations derived fromalkylamines such as ethylamine, diethylamine, triethylamine, andmixtures thereof, and the like). Typically, alkyl chains of C12-C16 arepreferred for lower wash temperatures (e.g. below about 50° C.) andC16-C18 alkyl chains are preferred for higher wash temperatures (e.g.above about 50° C.).

[0187] Other anionic surfactants useful for detersive purposes can alsobe included in the laundry detergent compositions of the presentinvention. Theses can include salts (including, for example, sodium,potassium, ammonium, and substituted ammonium salts such as mono- di-and triethanolamine salts) of soap, C8-C22 primary or secondaryalkanesulfonates, C8-C24 olefinsulfonates, sulfonated polycarboxylicacids prepared by sulfonation of the pyrolyzed product of alkaline earthmetal citrates, e.g., as described in British patent specification No.1,082,179, C8-C24 alkylpolyglycolethersulfates (containing up to 10moles of ethylene oxide); alkyl glycerol sulfonates, fatty acyl glycerolsulfonates, fatty oleyl glycerol sulfates, alkyl phenol ethylene oxideether sulfates, paraffin sulfonates, alkyl phosphates, isethionates suchas the acyl isethionates, N-acyl taurates, alkyl succinamates andsulfosuccinates, monoesters of sulfosuccinates (especially saturated andunsaturated C12-C18 monoesters) and diesters of sulfosuccinates(especially saturated and unsaturated C6-C12 diesters), acylsarcosinates, sulfates of alkylpolysaccharides such as the sulfates ofalkylpolyglucoside (the nonionic nonsulfated compounds being describedbelow), branched primary alkyl sulfates, and alkyl polyethoxycarboxylates such as those of the formula RO(CH2CH2O)k—CH2C00-M+ whereinR is a C8-C22 alkyl, k is an integer from 1 to 10, and M is a solublesalt forming cation. Resin acids and hydrogenated resin acids are alsosuitable, such as rosin, hydrogenated, rosin, and resin acids andhydrogenated resin acids present in or derived from tall oil.

[0188] Alkylbenzene sulfonates are highly preferred. Especiallypreferred are linear (straight-chain) alkyl benzene sulfonates (LAS)wherein the alkyl group preferably contains from 10 to 18 carbon atoms.

[0189] Further examples are described in “Surface Active Agents andDetergents” (Vol. I and II by Schwartz, Perrry and Berch). A variety ofsuch surfactants are also generally disclosed in U.S. Pat. No.3,929,678, (Column 23, line 58 through Column 29, line 23, hereinincorporated by reference).

[0190] When included therein, the laundry detergent compositions of thepresent invention typically comprise from about 1% to about 40%,preferably from about 3% to about 20% by weight of such anionicsurfactants.

[0191] The laundry detergent compositions of the present invention mayalso contain cationic, ampholytic, zwitterionic, and semi-polarsurfactants, as well as the nonionic and/or anionic surfactants otherthan those already described herein.

[0192] Cationic detersive surfactants suitable for use in the laundrydetergent compositions of the present invention are those having onelong-chain hydrocarbyl group. Examples of such cationic surfactantsinclude the ammonium surfactants such as alkyltrimethylammoniumhalogenides, and those surfactants having the formula:

[R2(OR3)y][R4(OR3)y]2R5N+X—

[0193] wherein R2 is an alkyl or alkyl benzyl group having from about 8to about 18 carbon atoms in the alkyl chain, each R3 is selected formthe group consisting of —CH2CH2—, —CH2CH(CH3)—, —CH2CH(CH20H)—,—CH2CH2CH2—, and mixtures thereof; each R4 is selected from the groupconsisting of C1-C4 alkyl, C1-C4 hydroxyalkyl, benzyl ring structuresformed by joining the two R4 groups, —CH2CHOHCHOHCOR6CHOHCH2OH, whereinR6 is any hexose or hexose polymer having a molecular weight less thanabout 1000, and hydrogen when y is not 0; R5 is the same as R4 or is analkyl chain, wherein the total number of carbon atoms or R2 plus R5 isnot more than about 18; each y is from 0 to about 10, and the sum of they values is from 0 to about 15; and X is any compatible anion.

[0194] Highly preferred cationic surfactants are the water solublequaternary ammonium compounds useful in the present composition havingthe formula:

R1R2R3R4N+X—  (i)

[0195] wherein R1 is C8-C16 alkyl, each of R2, R3 and R4 isindependently C1-C4 alkyl, C1-C4 hydroxy alkyl, benzyl, and —(C2H40)xHwhere x has a value from 2 to 5, and X is an anion. Not more than one ofR2, R3 or R4 should be benzyl.

[0196] The preferred alkyl chain length for R1 is C12-C15, particularlywhere the alkyl group is a mixture of chain lengths derived from coconutor palm kernel fat or is derived synthetically by olefin build up or OXOalcohols synthesis.

[0197] Preferred groups for R2R3 and R4 are methyl and hydroxyethylgroups and the anion X may be selected from halide, methosulphate,acetate and phosphate ions.

[0198] Examples of suitable quaternary ammonium compounds of formulae(i) for use herein are:

[0199] coconut trimethyl ammonium chloride or bromide;

[0200] coconut methyl dihydroxyethyl ammonium chloride or bromide;

[0201] decyl triethyl ammonium chloride;

[0202] decyl dimethyl hydroxyethyl ammonium chloride or bromide;

[0203] C12-15 dimethyl hydroxyethyl ammonium chloride or bromide;

[0204] coconut dimethyl hydroxyethyl ammonium chloride or bromide;

[0205] myristyl trimethyl ammonium methyl sulphate;

[0206] lauryl dimethyl benzyl ammonium chloride or bromide;

[0207] lauryl dimethyl (ethenoxy)4 ammonium chloride or bromide;

[0208] choline esters (compounds of formula (i) wherein R1 isCH2—CH2—O—C—C12-14 alkyl and R2R3R4 are methyl).

[0209] di-alkyl imidazolines [compounds of formula (i)].

[0210] Other cationic surfactants useful herein are also described inU.S. Pat. No. 4,228,044 and in EP 000 224.

[0211] When included therein, the laundry detergent compositions of thepresent invention typically comprise from 0.2% to about 25%, preferablyfrom about 1% to about 8% by weight of such cationic surfactants.

[0212] Ampholytic surfactants are also suitable for use in the laundrydetergent compositions of the present invention. These surfactants canbe broadly described as aliphatic derivatives of secondary or tertiaryamines, or aliphatic derivatives of heterocyclic secondary and tertiaryamines in which the aliphatic radical can be straight- orbranched-chain. One of the aliphatic substituents contains at leastabout 8 carbon atoms, typically from about 8 to about 18 carbon atoms,and at least one contains an anionic water-solubilizing group, e.g.carboxy, sulfonate, sulfate. See U.S. Pat. No. 3,929,678 (column 19,lines 18-35) for examples of ampholytic surfactants.

[0213] When included therein, the laundry detergent compositions of thepresent invention typically comprise from 0.2% to about 15%, preferablyfrom about 1% to about 10% by weight of such ampholytic surfactants.

[0214] Zwitterionic surfactants are also suitable for use in laundrydetergent compositions. These surfactants can be broadly described asderivatives of secondary and tertiary amines, derivatives ofheterocyclic secondary and tertiary amines, or derivatives of quaternaryammonium, quaternary phosphonium or tertiary sulfonium compounds. SeeU.S. Pat. No. 3,929,678 (column 19, line 38 through column 22, line 48)for examples of zwitterionic surfactants.

[0215] When included therein, the laundry detergent compositions of thepresent invention typically comprise from 0.2% to about 15%, preferablyfrom about 1% to about 10% by weight of such zwitterionic surfactants.

[0216] Semi-polar nonionic surfactants are a special category ofnonionic surfactants which include water-soluble amine oxides containingone alkyl moiety of from about 10 to about 18 carbon atoms and 2moieties selected from the group consisting of alkyl groups andhydroxyalkyl groups containing from about 1 to about 3 carbon atoms;water soluble phosphine oxides containing one alkyl moiety of from about10 to about 18 carbon atoms and 2 moieties selected from the groupconsisting of is alkyl groups and hydroxyalkyl groups containing fromabout 1 to about 3 carbon atoms; and water-soluble sulfoxides containingone alkyl moiety from about 10 to about 18 carbon atoms and a moietyselected from the group consisting of alkyl and hydroxyalkyl moieties offrom about 1 to about 3 carbon atoms.

[0217] Semi-polar nonionic detergent surfactants include the amine oxidesurfactants having the formula:

[0218] wherein R3 is an alkyl, hydroxyalkyl, or alkyl phenyl group ormixtures thereof containing from about 8 to about 22 carbon atoms; R4 isan alkylene or hydroxyalkylene group containing from about 2 to about 3carbon atoms or mixtures thereof; x is from 0 to about 3: and each R5 isan alkyl or hydroxyalkyl group containing from about 1 to about 3 carbonatoms or a polyethylene oxide group containing from about 1 to about 3ethylene oxide groups. The R5 groups can be attached to each other,e.g., through an oxygen or nitrogen atom, to form a ring structure.

[0219] These amine oxide surfactants in particular include C10-C18 alkyldimethyl amine oxides and C8-C12 alkoxy ethyl dihydroxy ethyl amineoxides.

[0220] When included therein, the laundry detergent compositions of thepresent invention typically comprise from 0.2% to about 15%, preferablyfrom about 1% to about 10% by weight of such semi-polar nonionicsurfactants.

[0221] Builder System

[0222] The compositions according to the present invention may furthercomprise a builder system. Any conventional builder system is suitablefor use herein including aluminosilicate materials, silicates,polycarboxylates and fatty acids, materials such as ethylenediaminetetraacetate, metal ion sequestrants such as aminopolyphosphonates,particularly ethylenediamine tetramethylene phosphonic acid anddiethylene triamine pentamethylenephosphonic acid. Though less preferredfor obvious environmental reasons, phosphate builders can also be usedherein.

[0223] Suitable builders can be an inorganic ion exchange material,commonly an inorganic hydrated aluminosilicate material, moreparticularly a hydrated synthetic zeolite such as hydrated zeolite A, X,B, HS or MAP.

[0224] Another suitable inorganic builder material is layered silicate,e.g. SKS-6 (Hoechst). SKS-6 is a crystalline layered silicate consistingof sodium silicate (Na2Si2O5).

[0225] Suitable polycarboxylates containing one carboxy group includelactic acid, glycolic acid and ether derivatives thereof as disclosed inBelgian Patent Nos. 831,368, 821,369 and 821,370. Polycarboxylatescontaining two carboxy groups include the water-soluble salts ofsuccinic acid, malonic acid, (ethylenedioxy) diacetic acid, maleic acid,diglycollic acid, tartaric acid, tartronic acid and fumaric acid, aswell as the ether carboxylates described in German Offenle-enschrift2,446,686, and 2,446,487, U.S. Pat. No. 3,935,257 and the sulfinylcarboxylates described in Belgian Patent No. 840,623. Polycarboxylatescontaining three carboxy groups include, in particular, water-solublecitrates, aconitrates and citraconates as well as succinate derivativessuch as the carboxymethyloxysuccinates described in British Patent No.1,379,241, lactoxysuccinates described in Netherlands Application7205873, and the oxypolycarboxylate materials such as2-oxa-1,1,3-propane tricarboxylates described in British Patent No.1,387,447.

[0226] Polycarboxylates containing four carboxy groups includeoxydisuccinates disclosed in British Patent No. 1,261,829,1,1,2,2,-ethane tetracarboxylates, 1,1,3,3-propane tetracarboxylatescontaining sulfo substituents include the sulfosuccinate derivativesdisclosed in British Patent Nos. 1,398,421 and 1,398,422 and in U.S.Pat. No. 3,936,448, and the sulfonated pyrolysed citrates described inBritish Patent No. 1,082,179, while polycarboxylates containingphosphone substituents are disclosed in British Patent No. 1,439,000.

[0227] Alicyclic and heterocyclic polycarboxylates includecyclopentane-cis,cis-cis-tetracarboxylates, cyclopentadienidepentacarboxylates, 2,3,4,5-tetrahydro-furan—cis, cis,cis-tetracarboxylates, 2,5-tetrahydro-furan-tetrahydro-furan-cis,discarboxylates, 2,2,5,5,-tetrahydrofuran—tetracarboxylates,1,2,3,4,5,6-hexane—hexacarboxylates and carboxymethyl derivatives ofpolyhydric alcohols such as sorbitol, mannitol and xylitol. Aromaticpolycarboxylates include mellitic acid, pyromellitic acid and thephthalic acid derivatives disclosed in British Patent No. 1,425,343.

[0228] Of the above, the preferred polycarboxylates arehydroxy-carboxylates containing up to three carboxy groups per molecule,more particularly citrates.

[0229] Preferred builder systems for use in the present compositionsinclude a mixture of a water-insoluble aluminosilicate builder such aszeolite A or of a layered silicate (SKS-6), and a water-solublecarboxylate chelating agent such as citric acid.

[0230] A suitable chelant for inclusion in the detergent composi-ions inaccordance with the invention is ethylenediamine-N,N′-disuccinic acid(EDDS) or the alkali metal, alkaline earth metal, ammonium, orsubstituted ammonium salts thereof, or mixtures thereof. Preferred EDDScompounds are the free acid form and the sodium or magnesium saltthereof. Examples of such preferred sodium salts of EDDS include Na2EDDSand Na4EDDS. Examples of such preferred magnesium salts of EDDS includeMgEDDS and Mg2EDDS. The magnesium salts are the most preferred forinclusion in compositions in accordance with the invention.

[0231] Preferred builder systems include a mixture of a water-insolublealuminosilicate builder such as zeolite A, and a water solublecarboxylate chelating agent such as citric acid.

[0232] Other builder materials that can form part of the builder systemfor use in granular compositions include inorganic materials such asalkali metal carbonates, bicarbonates, silicates, and organic materialssuch as the organic phosphonates, amino polyalkylene phosphonates andamino polycarboxylates.

[0233] Other suitable water-soluble organic salts are the homo- orco-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated form each other bynot more than two carbon atoms.

[0234] Polymers of this type are disclosed in GB-A-1,596,756. Examplesof such salts are polyacrylates of MW 2000-5000 and their copolymerswith maleic anhydride, such copolymers having a molecular weight of from20,000 to 70,000, especially about 40,000.

[0235] Detergency builder salts are normally included in amounts of from5% to 80% by weight of the composition. Preferred levels of builder forliquid detergents are from 5% to 30%.

[0236] Enzymes

[0237] Preferred detergent compositions, in addition to the enzymepreparation of the invention, comprise other enzyme(s) which providescleaning performance and/or fabric care benefits.

[0238] Such enzymes include proteases, lipases, cutinases, amylases,cellulases, peroxidases, oxidases (e.g. laccases).

[0239] Proteases: Any protease suitable for use in alkaline solutionscan be used. Suitable proteases include those of animal, vegetable ormicrobial origin. Microbial origin is preferred. Chemically orgenetically modified mutants are included. The protease may be a serineprotease, preferably an alkaline microbial protease or a trypsin-likeprotease. Examples of alkaline proteases are subtilisins, especiallythose derived from Bacillus, e.g., subtilisin Novo, subtilisinCarlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (describedin WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g. ofporcine or bovine origin) and the Fusarium protease described in WO89/06270.

[0240] Preferred commercially available protease enzymes include thosesold under the trade names Alcalase, Savinase, Primase, Durazym, andEsperase by Novo Nordisk A/S (Denmark), those sold under the tradenameMaxatase, Maxacal, Maxapem, Properase, Purafect and Purafect OXP byGenencor International, and those sold under the tradename Opticlean andOptimase by Solvay Enzymes. Protease enzymes may be incorporated intothe compositions in accordance with the invention at a level of from0.00001% to 2% of enzyme protein by weight of the composition,preferably at a level of from 0.0001% to 1% of enzyme protein by weightof the composition, more preferably at a level of from 0.001% to 0.5% ofenzyme protein by weight of the composition, even more preferably at Ealevel of from 0.01% to 0.2% of enzyme protein by weight of thecomposition.

[0241] Lipases: Any lipase suitable for use in alkaline solutions can beused. Suitable lipases include those of bacterial or fungal origin.Chemically or genetically modified mutants are included.

[0242] Examples of useful lipases include a Humicola lanuginosa lipase,e.g., as described in EP 258 068 and EP 305 216, a Rhizomucor mieheilipase, e.g., as described in EP 238 023, a Candida lipase, such as a C.antarctica lipase, e.g., the C. antarctica lipase A or B described inEP: 214 761, a Pseudomonas lipase such as a P. alcaligenes and P.pseudoalcaligenes lipase, e.g., as described in EP 218 272, a P. cepacialipase, e.g., as described in EP 331 376, a P. stutzeri lipase, e.g., asdisclosed in GB 1,372,034, a P. fluorescens lipase, a Bacillus lipase,e.g., a B. subtilis lipase (Dartois et al., (1993), Biochemica etBiophysica acta 1131, 253-260), a B. stearothermophilus lipase (JP64/744992) and a B. pumilus lipase (WO 91/16422).

[0243] Furthermore, a number of cloned lipases may be useful, includingthe Penicillium camembertii lipase described by Yamaguchi et al.,(1991), Gene 103, 61-67), the Geotricum candidum lipase (Schimada, Y. etal., (1989), J. Biochem., 106, 383-388), and various Rhizopus lipasessuch as a R. delemar lipase (Hass, M. J. et al., (1991), Gene 109,117-113), a R. niveus lipase (Kugimiya et al., (1992), Biosci. Biotech.Biochem. 56, 716-719) and a R. oryzae lipase.

[0244] Other types of lipolytic enzymes such as cutinases may also beuseful, e.g., a cutinase derived from Pseudomonas mendocina as describedin WO 88/09367, or a cutinase derived from Fusarium solani pisi (e.g.described in WO 90/09446).

[0245] Especially suitable lipases are lipases such as M1 LipaseTM, LumafastTM and LipomaxTM (Genencor), LipolaseTM and Lipolase UltraTM (NovoNordisk A/S), and Lipase P “Amano” (Amano Pharmaceutical Co. Ltd.).

[0246] The lipases are normally incorporated in the detergentcomposition at a level of from 0.00001% to 2% of enzyme protein byweight of the composition, preferably at a level of from 0.0001% to 1%of enzyme protein by weight of the composition, more preferably at alevel of from 0.001% to 0.5% of enzyme protein by weight of thecomposition, even more preferably at a level of from 0.01% to 0.2% ofenzyme protein by weight of the composition.

[0247] Amylases: Any amylase (a and/or b) suitable for use in alkalinesolutions can be used. Suitable amylases include those of bacterial orfungal origin. Chemically or genetically modified mutants are included.Amylases include, for example, a-amylases obtained from a special strainof B. licheniformis, described in more detail in GB 1,296,839.Commercially available amylases are DuramylTM, TermamylTM, FungamylTMand BANTM (available from Novo Nordisk A/S) and RapidaseTM and MaxamylPTM (available from Genencor).

[0248] The amylases are normally incorporated in the detergentcomposition at a level of from 0.00001% to 2% of enzyme protein byweight of the composition, preferably at a level of from 0.0001% to 1%of enzyme protein by weight of the composition, more preferably at alevel of from 0.001% to 0.5% of enzyme protein by weight of thecomposition, even more preferably at a level of from 0.01% to 0.2% ofenzyme protein by weight of the composition.

[0249] Cellulases: Any cellulase suitable for use in alkaline solutionscan be used. Suitable cellulases include those of bacterial or fungalorigin. Chemically or genetically modified mutants are included.Suitable cellulases are disclosed in U.S. Pat. No. 4,435,307, whichdiscloses fungal cellulases produced from Humicola insolens. Especiallysuitable cellulases are the 15 cellulases having color care benefits.Examples of such cellulases are cellulases described in European patentapplication No. 0 495 257 and the endoglucanase of the presentinvention.

[0250] Commercially available cellulases include CelluzymeTM produced bya strain of Humicola insolens, (Novo Nordisk A/S), and KAC-500(B)TM (KaoCorporation).

[0251] Cellulases are normally incorporated in the detergent compositionat a level of from 0.00001% to 2% of enzyme protein by weight of thecomposition, preferably at a level of from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level of from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level of from 0.01% to 0.2% of enzyme protein by weightof the composition.

[0252] Peroxidases/Oxidases: Peroxidase enzymes are used in combinationwith hydrogen peroxide or a source thereof (e.g. a percarbonate,perborate or persulfate). Oxidase enzymes are used in combination withoxygen. Both types of enzymes are used for “solution bleaching”, i.e. toprevent transfer of a textile dye from a dyed fabric to another fabricwhen said fabrics are washed together in a wash liquor, preferablytogether with an enhancing agent as described in e.g. WO 94/12621 and WO95/01426. Suitable peroxidases/oxidases include those of plant,bacterial or fungal origin. Chemically or genetically modified mutantsare included.

[0253] Peroxidase and/or oxidase enzymes are normally incorporated inthe detergent composition at a level of from 0.00001% to 2% of enzymeprotein by weight of the composition, preferably at a level of from0.0001% to 1% of enzyme protein by weight of the composition, morepreferably at a level of from 0.001% to 0.5% of enzyme protein by weightof the composition, even more preferably at a level of from 0.01% to0.2% of enzyme protein by weight of the composition.

[0254] Mixtures of the above mentioned enzymes are encompassed herein,in particular a mixture of a protease, an amylase, a lipase and/or acellulase.

[0255] The enzyme of the invention, or any other enzyme incorporated inthe detergent composition, is normally incorporated in the detergentcomposition at a level from 0.00001% to 2% of enzyme protein by weightof the composition, preferably at a level from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level from 0.01% to 0.2% of enzyme protein by weight ofthe composition.

[0256] Bleaching Agents

[0257] Additional optional detergent ingredients that can be included inthe detergent compositions of the present invention include bleachingagents such as PB1, PB4 and percarbonate with a particle size of 400-800microns. These bleaching agent components can include one or more oxygenbleaching agents and, depending upon the bleaching agent chosen, one ormore bleach activators. When present oxygen bleaching compounds willtypically be present at levels of from about 1% to about 25%. Ingeneral, bleaching compounds are optional added components in non-liquidformulations, e.g. granular detergents.

[0258] The bleaching agent component for use herein can be any of thebleaching agents useful for detergent compositions including oxygenbleaches as well as others known in the art.

[0259] The bleaching agent suitable for the present invention can be anactivated or non-activated bleaching agent.

[0260] One category of oxygen bleaching agent that can be usedencompasses percarboxylic acid bleaching agents and salts thereof.Suitable examples of this class of agents include magnesiummonoperoxyphthalate hexahydrate, the magnesium salt of meta-chloroperbenzoic acid, 4-nonylamino-4-oxoperoxybutyric acid anddiperoxydodecanedioic acid. Such bleaching agents are disclosed in U.S.Pat. No. 4,483,781, U.S. Pat. No. 740,446, EP 0 133 354 and U.S. Pat.No. 4,412,934. Highly preferred bleaching agents also include6-nonylamino-6-oxoperoxycaproic acid as described in U.S. Pat. No.4,634,551.

[0261] Another category of bleaching agents that can be used encompassesthe halogen bleaching agents. Examples of hypohalite bleaching agents,for example, include trichloro isocyanuric acid and the sodium andpotassium dichloroisocyanurates and N-chloro and N-bromo alkanesulphonamides. Such materials are normally added at 0.5-10% by weight ofthe finished product, preferably 1-5% by weight.

[0262] The hydrogen peroxide releasing agents can be used in combinationwith bleach activators such as tetra-acetylethylenediamine (TAED),nonanoyloxybenzenesulfonate (NOBS, described in U.S. Pat. No.4,412,934), 3,5-trimethyl-hexsanoloxybenzenesulfonate (ISONOBS,described in EP 120 591) or pentaacetylglucose (PAG), which areperhydrolyzed to form a peracid as the active bleaching species, leadingto improved bleaching effect. In addition, very suitable are the bleachactivators C8(6-octanamido-caproyl) oxybenzene-sulfonate,C9(6-nonanamido caproyl) oxybenzenesulfonate and C10 (6-decanamidocaproyl) oxybenzenesulfonate or mixtures thereof. Also suitableactivators are acylated citrate esters such as disclosed in EuropeanPatent Application No. 91870207.7.

[0263] Useful bleaching agents, including peroxyacids and bleachingsystems comprising bleach activators and peroxygen bleaching compoundsfor use in cleaning compositions according to the invention aredescribed in application U.S. Ser. No. 08/136,626.

[0264] The hydrogen peroxide may also be present by adding an enzymaticsystem (i.e. an enzyme and a substrate therefore) which is capable ofgeneration of hydrogen peroxide at the beginning or during the washingand/or rinsing process. Such enzymatic systems are disclosed in EuropeanPatent Application EP 0 537 381.

[0265] Bleaching agents other than oxygen bleaching agents are alsoknown in the art and can be utilized herein. One type of non-oxygenbleaching agent of particular interest includes photoactivated bleachingagents such as the sulfonated zinc and/or aluminium phthalocyanines.These materials can be deposited upon the substrate during the washingprocess. Upon irradiation with light, in the presence of oxygen, such asby hanging clothes out to dry in the daylight, the sulfonated zincphthalocyanine is activated and, consequently, the substrate isbleached. Preferred zinc phthalocyanine and a photoactivated bleachingprocess are described in U.S. Pat. No. 4,033,718. Typically, detergentcomposition will contain about 0.025% to about 1.25%, by weight, ofsulfonated zinc phthalocyanine.

[0266] Bleaching agents may also comprise a manganese catalyst. Themanganese catalyst may, e.g., be one of the compounds described in“Efficient manganese catalysts for low-temperature bleaching”, Nature369, 1994, pp. 637-639.

[0267] Suds Suppressors

[0268] Another optional ingredient is a suds suppressor, exemplified bysilicones, and silica-silicone mixtures. Silicones can generally berepresented by alkylated polysiloxane materials, while silica isnormally used in finely divided forms exemplified by silica aerogels andxerogels and hydrophobic silicas of various types. Theses materials canbe incorporated as particulates, in which the suds suppressor isadvantageously releasably incorporated in a water-soluble orwater-dispersible, substantially non surface-active detergentimpermeable carrier. Alternatively the suds suppressor can be dissolvedor dispersed in a liquid carrier and applied by spraying on to one ormore of the other components.

[0269] A preferred silicone suds controlling agent is disclosed in U.S.Pat. No. 3,933,672. Other particularly useful suds suppressors are theself-emulsifying silicone suds suppressors, described in German PatentApplication DTOS 2,646,126. An example of such a compound is DC-544,commercially available form Dow Corning, which is a siloxane-glycolcopolymer. Especially preferred suds controlling agent are the sudssuppressor system comprising a mixture of silicone oils and2-alkyl-alkanols. Suitable 2-alkyl-alkanols are 2-butyl-octanol whichare commercially available under the trade name Isofol 12 R.

[0270] Such suds suppressor system are described in European PatentApplication EP 0 593 841.

[0271] Especially preferred silicone suds: controlling agents aredescribed in European Patent Application No. 92201649.8. Saidcompositions can comprise a silicone/silica mixture in combination withfumed nonporous silica such as AerosilR.

[0272] The suds suppressors described above are normally employed atlevels of from 0.001% to 2% by weight of the composition, preferablyfrom 0.01% to 1% by weight.

[0273] Other Components

[0274] Other components used in detergent compositions may be employedsuch as soil-suspending agents, soil-releasing agents, opticalbrighteners, abrasives, bactericides, tarnish inhibitors, coloringagents, and/or encapsulated or nonencapsulated perfumes.

[0275] Especially suitable encapsulating materials are water solublecapsules which consist of a matrix of polysaccharide and polyhydroxycompounds such as described in GB 1,464,616.

[0276] Other suitable water soluble encapsulating materials comprisedextrins derived from ungelatinized starch acid esters of substituteddicarboxylic acids such as described in U.S. Pat. No. 3,455,838. Theseacid-ester dextrins are, preferably, prepared from such starches as waxymaize, waxy sorghum, sago, tapioca and potato. Suitable examples of saidencapsulation materials include N-Lok manufactured by National Starch.The N-Lok encapsulating material consists of a modified maize starch andglucose. The starch is modified by adding monofunctional substitutedgroups such as octenyl succinic acid anhydride.

[0277] Antiredeposition and soil suspension agents suitable hereininclude cellulose derivatives such as methylcellulose,carboxymethylcellulose and hydroxyethylcellulose, and homo- orco-polymeric polycarboxylic acids or their salts. Polymers of this typeinclude the polyacrylates and maleic anhydride-acrylic acid copolymerspreviously mentioned as builders, as well as copolymers of maleicanhydride with ethylene, methylvinyl ether or methacrylic acid, themaleic anhydride constituting at least 20 mole percent of the copolymer.These materials are normally used at levels of from 0.5% to 10% byweight, more preferably form 0.75% to 8%, most preferably from 1% to 6%by weight of the composition.

[0278] Preferred optical brighteners are anionic in character, examplesof which are disodium 4,4′-bis-(2-diethanolamino-4-anilino -s-triazin-6-ylamino)stilbene-2:2′-disulphonate, disodium 4,-4′-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino-stilbene-2:2′-disulphonate,disodium4,4′bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2:2′-disulphonate,monosodium 4′,4″bis-(2,4-dianilino-s-tri-azin-6ylamino)stilbene-2-sulphonate, disodium4,4′-bis-(2-anillino-4-(N-methyl-N-2-hydroxethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphate, di-sodium4,4′-bis-(4-phenyl-2,1,3-triazol-2-yl)-stilbene-2,2′ disulphonate,di-so-dium 4,4′bis(2-anilino-4-(1-methyl-2-hydroxyethylamino)-s-triazin-6-ylamino)stilbene-2,2′disulphonate, sodium2(stilbyl-4″-(naphtho-1′,2′:4,5)-1,2,3,-triazole-2″-sulphonate and4,4′-bis (2-sulphostyryl)biphenyl.

[0279] Other useful polymeric materials are the polyethylene glycols,particularly those of molecular weight 1000-10000, more particularly2000 to 8000 and most preferably about 4000. These are used at levels offrom 0.20% to 5% more preferably from 0.25% to 2.5% by weight. Thesepolymers and the previously mentioned homo- or co-polymericpoly-carboxylate salts are valuable for improving whiteness maintenance,fabric ash deposition, and cleaning performance on clay, proteinaceousand oxidizable soils in the presence of transition metal impurities.

[0280] Soil release agents useful in compositions of the resentinvention are conventionally copolymers or terpolymers f terephthalicacid with ethylene glycol and/or propylene lycol units in variousarrangements. Examples of such polymers are disclosed in U.S. Pat. Nos.4,116,885 and 4,711,730 and EP 0 272 033. A particular preferred polymerin accordance with EP 0 272 033 has the formula:

(CH3)(PEG)43)0.75(POH)0.25[T-PO)(T-PEG)0.4]T(POH)0.25((PEG)43CH3)0.75

[0281] where PEG is —(OC2H4)O—, PO is (OC3H6O) and T is (pOOC6H4CO).

[0282] Also very useful are modified polyesters as random copolymers ofdimethyl terephthalate, dimethyl sulfoisophthalate, ethylene glycol and1,2-propanediol, the end groups consisting primarily of sulphobenzoateand secondarily of mono esters of ethylene glycol and/or1,2-propanediol. The target is to obtain a polymer capped at both end bysulphobenzoate groups, “primarily”, in the present context most of saidcopolymers herein will be endcapped by sulphobenzoate groups. However,some copolymers will be less than fully capped, and therefore their endgroups may consist of monoester of ethylene glycol and/or1,2-propanediol, thereof consist “secondarily” of such species.

[0283] The selected polyesters herein contain about 46% by weight ofdimethyl terephthalic acid, about 16% by weight of 1,2-propanediol,about 10% by weight ethylene glycol, about 13% by weight of dimethylsulfobenzoic acid and about 15% by weight of sulfoisophthalic acid, andhave a molecular weight of about 3.000. The polyesters and their methodof preparation are described in detail in EP 311 342.

[0284] Softening Agents

[0285] Fabric softening agents can also be incorporated into laundrydetergent compositions in accordance with the present invention. Theseagents may be inorganic or organic in type. Inorganic softening agentsare exemplified by the smectite clays disclosed in GB-A-1 400898 and inU.S. Pat. No. 5,019,292. Organic fabric softening agents include thewater insoluble tertiary amines as disclosed in GB-A1 514 276 and EP 0011 340 and their combination with mono C12-C14 quaternary ammoniumsalts are disclosed in Eβ-B-0 026 528 and di-long-chain amides asdisclosed in EP 0 242 919. Other useful organic ingredients of fabricsoftening systems include high molecular weight polyethylene oxidematerials as disclosed in EP 0 299 575 and 0 313 146.

[0286] Levels of smectite clay are normally in the range from 5% to 15%,more preferably from 8% to 12% by weight, with the material being addedas a dry mixed component to the remainder of the formulation. Organicfabric softening agents such as the water-insoluble tertiary amines ordilong chain amide materials are incorporated at levels of from 0.5% to5% by weight, normally from 1% to 3% by weight whilst the high molecularweight polyethylene oxide materials and the water soluble cationicmaterials are added at levels of from 0.1% to 2%, normally from 0.15% to1.5% by weight. These materials are normally added to the spray driedportion of the composition, although in some instances it may be moreconvenient to add them as a dry mixed particulate, or spray them asmolten liquid on to other solid components of the composition.

[0287] Polymeric Dye-transfer Inhibiting Agents

[0288] The detergent compositions according to the present invention mayalso comprise from 0.001% to 10%, preferably from 0.01% to 2%, morepreferably form 0.05% to 1% by weight of polymeric dye- transferinhibiting agents. Said polymeric dye-transfer inhibiting agents arenormally incorporated into detergent compositions in order to inhibitthe transfer of dyes from colored fabrics onto fabrics washed therewith.These polymers have the ability of complexing or adsorbing the fugitivedyes washed out of dyed fabrics before the dyes have the opportunity tobecome attached to other articles in the wash.

[0289] Especially suitable polymeric dye-transfer inhibiting agents arepolyamine N-oxide polymers, copolymers of N-vinyl-pyrrolidone andN-vinylimidazole, polyvinylpyrrolidone polymers, polyvinyloxazolidonesand polyvinylimidazoles or mixtures thereof.

[0290] Addition of such polymers also enhances the performance of theenzymes according the invention.

[0291] The detergent composition according to the invention can be inliquid, paste, gels, bars or granular forms.

[0292] Non-dusting granulates may be produced, e.g., as disclosed inU.S. Pat. Nos. 4,106,991 and 4,661,452 (both to Novo Industri A/S) andmay optionally be coated by methods known in the art. Examples of waxycoating materials are poly(ethylene oxide) products (polyethyleneglycol,PEG) with mean molecular weights of 1000 to 20000; ethoxylatednonylphenols having from 16 to 50 ethylene oxide units; ethoxylatedfatty alcohols in which the alcohol contains from 12 to 20 carbon atomsand in which there are 15 to 80 ethylene oxide units; fatty alcohols;fatty acids; and mono- and di- and triglycerides of fatty acids.Examples of film-forming coating materials suitable for application byfluid bed techniques are given in GB 1483591.

[0293] Granular compositions according to the present invention can alsobe in “compact form”, i.e. they may have a relatively higher densitythan conventional granular detergents, i.e. form 550 to 950 g/l; in suchcase, the granular detergent compositions according to the presentinvention will contain a lower amount of “Inorganic filler salt”,compared to conventional granular detergents; typical filler salts arealkaline earth metal salts of sulphates and chlorides, typically sodiumsulphate; “Compact” detergent typically comprise not more than 10%filler salt. The liquid compositions according to the present inventioncan also be in “concentrated form”, in such case, the liquid detergentcompositions according to the present invention will contain a loweramount of water, compared to conventional liquid detergents. Typically,the water content of the concentrated liquid detergent is less than 30%,more preferably less than 20%, most preferably less than 10% by weightof the detergent compositions.

[0294] The compositions of the invention may for example, be formulatedas hand and machine laundry detergent compositions including laundryadditive compositions and compositions suitable for use in thepretreatment of stained fabrics, rinse added fabric softenercompositions, and compositions for use in general household hard surfacecleaning operations and dishwashing operations.

[0295] The following examples are meant to exemplify compositions forthe present invention, but are not necessarily meant to limit orotherwise define the scope of the invention.

[0296] In the detergent compositions, the abbreviated componentidentifications have the following meanings:

[0297] LAS: Sodium linear C12 alkyl benzene sulphonate

[0298] TAS: Sodium tallow alkyl sulphate

[0299] XYAS: Sodium C1X-C1Y alkyl sulfate

[0300] SS: Secondary soap surfactant of formula 2-butyl octanoic acid

[0301] 25EY: A C12-C15 predominantly linear primary alcohol condensedwith an average of Y moles of ethylene oxide

[0302] 45EY: A C14-C15 predominantly linear primary alcohol condensedwith an average of Y moles of ethylene oxide

[0303] XYEZS: C1X-C1Y sodium alkyl sulfate condensed with an average ofZ moles of ethylene oxide per mole

[0304] Nonionic: C13-C15 mixed ethoxylated/propoxylated fatty alcoholwith an average degree of ethoxylation of 3.8 and an average degree ofpropoxylation of 4.5 sold under the tradename Plurafax LF404 by BASFGmbh

[0305] CFAA: C12-C14 alkyl N-methyl glucamide

[0306] TFAA: C16-C18 alkyl N-methyl glucamide

[0307] Silicate: Amorphous Sodium Silicate (SiC)2:Na2O ratio=2.0)

[0308] NaSKS-6: Crystalline layered silicate of formula d-Na2Si2O5

[0309] Carbonate: Anhydrous sodium carbonate

[0310] Phosphate: Sodium tripolyphosphate

[0311] MA/AA: Copolymer of 1:4 maleic/acrylic acid, average molecularweight about 80,000

[0312] Polyacrylate: Polyacrylate homopolymer with an average molecularweight of 8,000 sold under the tradename PA30 by BASF Gmbh

[0313] Zeolite A: Hydrated Sodium Aluminosilicate of formulaNa12(AlO2SiO2)12. 27H2O having a primary particle size in the range from1 to 10 micrometers

[0314] Citrate: Tri-sodium citrate dihydrate

[0315] Citric: Citric Acid

[0316] Perborate: Anhydrous sodium perborate monohydrate bleach,empirical formula NaBO2.H2O2

[0317] PB4: Anhydrous sodium perborate tetrahydrate

[0318] Percarbonate: Anhydrous sodium percarbonate bleach of empiricalformula 2Na2CO3.3H2O2

[0319] TAED: Tetraacetyl ethylene diamine

[0320] CMC: Sodium carboxymethyl cellulose

[0321] DETPMP: Diethylene triamine penta (methylene phosphonic acid),marketed by Monsanto under the Tradename Dequest 2060

[0322] PVP: Polyvinylpyrrolidone polymer

[0323] EDDS: Ethylenediamine-N, N′-disuccinic acid, [S,S] isomer in theform of the sodium salt

[0324] Suds Suppressor: 25% paraffin wax Mpt 50° C., 17% hydrophobicsilica, 58% paraffin oil

[0325] Granular Suds suppressor: 12% Silicone/silica, 18% stearylalcohol, 70% starch in granular form

[0326] Sulphate: Anhydrous sodium sulphate

[0327] HMWPEO: High molecular weight polyethylene oxide

[0328] TAE 25: Tallow alcohol ethoxylate (25)

DETERGENT EXAMPLE I

[0329] A granular fabric cleaning composition in accordance with theinvention may be prepared as follows: Sodium linear C12 alkyl 6.5benzene sulfonate Sodium sulfate 15.0 Zeolite A 26.0 Sodiumnitrilotriacetate 5.0 Enzyme of the invention 0.1 PVP 0.5 TAED 3.0 Boricacid 4.0 Perborate 18.0 Phenol sulphonate 0.1 Minors Up to 100

DETERGENT EXAMPLE II

[0330] A compact granular fabric cleaning composition (density 800 g/l)in accord with the invention may be prepared as follows: follows: 45AS8.0 25E3S 2.0 25E5 3.0 25E3 3.0 TFAA 2.5 Zeolite A 17.0 NaSKS-6 12.0Citric acid 3.0 Carbonate 7.0 MA/AA 5.0 CMC 0.4 Enzyme of the invention0.1 TAED 6.0 Percarbonate 22.0 EDDS 0.3 Granular suds suppressor 3.5water/minors Up to 100%

DETERGENT EXAMPLE III

[0331] Granular fabric cleaning compositions in accordance with theinvention which are especially useful in the laundering of colouredfabrics were prepared as follows: LAS 10.7 — TAS 2.4 — TFAA — 4.0 45AS3.1 10.0 45E7 4.0 — 25E3S — 3.0 68E11 1.8 — 25E5 — 8.0 Citrate 15.0 7.0Carbonate — 10 Citric acid 2.5 3.0 Zeolite A 32.1 25.0 Na-SKS-6 — 9.0MA/AA 5.0 5.0 DETPMP 0.2 0.8 Enzyme of the invention 0.10 0.05 Silicate2.5 — Sulphate 5.2 3.0 PVP 0.5 — Poly (4-vinylpyridine)-N- — 0.2Oxide/copolymer of vinyl- imidazole and vinyl- pyrrolidone Perborate 1.0— Phenol sulfonate 0.2 — Water/Minors Up to 100%

DETERGENT EXAMPLE IV

[0332] Granular fabric cleaning compositions in accordance with theinvention which provide “Softening through the wash” capability may beprepared as follows: 45AS — 10.0 LAS 7.6 — 68AS 1.3 — 45E7 4.0 — 25E3 —5.0 Coco-alkyl-dimethyl hydroxy- 1.4 1.0 ethyl ammonium chloride Citrate5.0 3.0 Na-SKS-G — 11.0 Zeolite A 15.0 15.0 MA/AA 4.0 4.0 DETPMP 0.4 0.4Perborate 15.0 — Percarbonate — 15.0 TAED 5.0 5.0 Smectite clay 10.010.0 HMWPEO — 0.1 Enzyme of the invention 0.10 0.05 Silicate 3.0 5.0Carbonate 10.0 10.0 Granular suds suppressor 1.0 4.0 CMC 0.2 0.1Water/Minors Up to 100%

DETERGENT EXAMPLE V

[0333] Heavy duty liquid fabric cleaning compositions in accordance withthe invention may be prepared as follows: I II LAS acid form — 25.0Citric acid 5.0 2.0 25AS acid form 8.0 — 25AE2S acid form 3.0 — 25AE78.0 — CFAA 5 — DETPMP 1.0 1.0 Fatty acid 8 — Oleic acid — 1.0 Ethanol4.0 5.0 Propanediol 2.0 6.0 Enzyme of the invention 0.10 0.05 Coco-alkyldimethyl — 3.0 hydroxy ethyl ammonium chloride Smectite clay — 5.0 PVP2.0 — Water/Minors Up to 100%

[0334] Textile Applications

[0335] In another embodiment, the present: invention relates to use ofthe endoglucanase of the invention in the bio-polishing process.Bio-Polishing is a specific treatment of the yarn surface which improvesfabric quality with respect to handle and appearance without loss offabric wettability. The most important effects of Bio-Polishing can becharacterized by less fuzz and pilling, increased gloss/luster, improvedfabric handle, increased durable softness and altered water absorbency.Bio-Polishing usually takes place in the wet processing of themanufacture of knitted and woven fabrics. Wet processing comprises suchsteps as e.g. desizing, scouring, bleaching, washing, dying/printing andfinishing. During each of these steps, the fabric is more or lesssubjected to mechanical action. In general, after the textiles have beenknitted or woven, the fabric proceeds to a desizing stage, followed by ascouring stage, etc. Desizing is the act of removing size from textiles.Prior to weaving on mechanical looms, warp yarns are often coated withsize starch or starch derivatives in order to increase their tensilestrength. After weaving, the size coating must be removed before furtherprocessing the fabric in order to ensure Ea homogeneous and wash-proofresult. It is known that in order to achieve the effects ofBio-Polishing, a combination of cellulytic and mechanical action isrequired. It is also known that “super-softness” is achievable when thetreatment with a cellulase is combined with a conventional treatmentwith softening agents. It is contemplated that use of the endoglucanaseof the invention for bio-polishing of cellulosic fabrics isadvantageous, e.g. a more thorough polishing can be achieved.Bio-polishing may be obtained by applying the method described e.g. inWO 93/20278.

[0336] Stone-Washing

[0337] It is known to provide a “stone-washed” look (localized abrasionof the color) in dyed fabric, especially in denim fabric or jeans,either by washing the denim or jeans made from such fabric in thepresence of pumice stones to provide the desired localized lightening ofthe color of the fabric or by treating the fabric enzymatically, inparticular with cellulytic enzymes. The treatment with an endoglucanaseof the present invention may be carried out either alone such asdisclosed in U.S. Pat. No. 4,832,864, together with a smaller amount ofpumice than required in the traditional process, or together withperlite such as disclosed in WO 95/09225.

[0338] Pulp and Paper Applications

[0339] In the papermaking pulp industry, the endoglucanase of thepresent invention may be applied advantageously e.g. as follows:

[0340] For debarking: pretreatment with the endoglucanase may degradethe cambium layer prior to debarking in mechanical drums resulting inadvantageous energy savings.

[0341] For defibration: treatment of a material containing cellulosicfibers with the endoglucanase prior to refining or beating may result inreduction of the energy consumption due to the hydrolysing effect of thecellulase on the interfibre surfaces. Use of the endoglucanase mayresult in improved energy savings as compared to the use of knownenzymes, since it is believed that the enzyme composition of theinvention may possess a higher ability to penetrate fibre walls.

[0342] For fibre modification, i.e. improvement of fibre propertieswhere partial hydrolysis across the fibre wall is needed which requiresdeeper penetrating enzymes (e.g. in order to make coarse fibers moreflexible). Deep treatment of fibers has so far not been possible forhigh yield pulps e.g. mechanical pulps or mixtures of recycled pulps.This has been ascribed to the nature of the fibre wall structure thatprevents the passage of enzyme molecules due to physical restriction ofthe pore matrix of the fibre wall. It is contemplated that the presentendoglucanase is capable of penetrating into the fibre wall.

[0343] For drainage improvement. The drainability of papermaking pulpsmay be improved by treatment of the pulp with hydrolysing enzymes, e.g.cellulases. Use of the present endoglucanase may be more effective, e.g.result in a higher degree of loosening bundles of strongly hydratedmicro-fibrils in the fines fraction (consisting of fibre debris) thatlimits the rate of drainage by blocking hollow spaces between fibers andin the wire mesh of the paper machine. The Canadian standard freeness(CSF) increases and the Schopper-Riegler drainage index decreases whenpulp in subjected to cellulase treatment, see e.g. U.S. Pat. No.4,923,565; TAPPI T227, SCAN C19:65.ence.

[0344] For inter fibre bonding. Hydrolytic enzymes are applied in themanufacture of papermaking pulps for improving the inter fibre bonding.The enzymes rinse the fibre surfaces for impurities e.g. cellulosicdebris, thus enhancing the area of exposed cellulose with attachment tothe fibre wall, thus improving the fibre-to-fibre hydrogen bindingcapacity. This process is also referred to as dehornification. Paper andboard produced with a cellulase containing enzyme preparation may havean improved strength or a reduced grammage, a smoother surface and animproved printability.

[0345] For enzymatic deinking. Partial hydrolysis of recycled paperduring or upon pulping by use of hydrolysing enzymes such as cellulasesare known to facilitate the removal and agglomeration of ink particles.Use of the present endoglucanse may give a more effective loosening ofink from the surface structure due to a better penetration of the enzymemolecules into the fibrillar matrix of the fibre wall, thus softeningthe surface whereby ink particles are effectively loosened. Theagglomeration of loosened ink particles are also improved, due to a moreefficient hydrolysis of cellulosic fragments found attached to inkparticles originating from the fibres.

[0346] The treatment of lignocellulosic pulp may, e.g., be performed asdescribed in WO 91/14819, WO 91/14822, WO 92/17573 and WO 92/18688.

[0347] Degradation of Plant Material

[0348] In yet another embodiment, the present invention relates to useof the endoglucanase and/or enzyme preparation according to theinvention for degradation of plant material e.g. cell walls.

[0349] It is contemplated that the novel endoglucanase and/or enzymepreparation of the invention is useful in the preparation of wine, fruitor vegetable juice in order to increase yield. Endoglucanases accordingto the invention may also be applied for enzymatic hydrolysis of variousplant cell-wall derived materials or waste materials, e.g. agriculturalresidues such as wheat-straw, corn cobs, whole corn plants, nut shells,grass, vegetable hulls, bean hulls, spent grains, sugar beet pulp, andthe like. The plant material may be degraded in order to improvedifferent kinds of processing, facilitate purification or extraction ofother components like purification of beta-glucan or beta-glucanoligomers from cereals, improve the feed value, decrease the waterbinding capacity, improve the degradability in waste water plants,improve the conversion of e.g. grass and corn to ensilage, etc.

EXAMPLES

[0350] The invention is further illustrated in the following exampleswhich are not intended to be in any way limiting to the scope of theinvention as claimed.

[0351] Materials and Methods

[0352] Cellulolytic Activity

[0353] The cellulase variants of the invention show improvedperformance. Some of the variants may show improved performance withrespect to increased catalytic activity.

[0354] In the context of this invention, cellulase activity can beexpressed in S-CEVU. Cellulolytic enzymes hydrolyse CMC, therebyincreasing the viscosity of the incubation mixture. The resultingreduction in viscosity may be determined by a vibration viscosimeter(e.g. MIVI 3000 from Sofraser, France).

[0355] Determination of the cellulolytic activity, measured in terms ofS-CEVU, may be determined according to the following analysis method(assay): The S-CEVU assay quantifies the amount of catalytic activitypresent in the sample by measuring the ability of the sample to reducethe viscosity of a solution of carboxy-methylcellulose (CMC). The assayis carried out at 40° C.; pH 7.5; 0.1M phosphate buffer; time 30 min;using a relative enzyme standard for reducing the viscosity of theCMC(carboxymethylcellulose Hercules 7 LFD) substrate; enzymeconcentration approx. 0.15 S-CEVU/ml. The arch standard is defined to8200 S-CEVU/g.

Example 1

[0356] Preparation of Cellulase Variants

[0357] Based on the disclosed sequence alignment (Table 1) and computermodeling method, position 119 was identified as a particular point ofinterest for making cellulase variants. Position 119 (cellulasenumbering) is located within 3 Å from the substrate. In position 119 thewild-type Humicola insolens cellulase holds a histidine residue (H),whereas the wild-type Thielavia terrestris cellulase holds a glutamineresidue (Q).

[0358] In this experiment, histidine was substituted for glutamine inthe Thielavia terrestris cellulase (thereby obtaining the cellulasevariant Thielavia terrestris/Q119H). The variant obtained was tested forspecific activity.

[0359] All Humicola insolens variants are, unless otherwise stated,constructed by application of the Chameleon™ Double-stranded,site-directed Mutagenesis kit, from Stratagene. The following syntheticoligo-nucleotide were used as selection primer: S/MGAATGACTTGGTTGACGCGTCACCAGTCAC, or M/S GAATGACTTGGTTGAGTACTCACCAGTCAC.

[0360] S/M replaces the ScaI site in the beta-lactamase gene of theplasmid with a M1uI site and M/S does the reverse. The later is used tointroduce secondary mutations in variants generated by the firstselection primer.

[0361] For construction of Thielavia terrestis cellulase variants, theThielavia terrestis EG V cellulase cDNA obtainable from the plasmiddeposited as DSM 10811 was used. DSM 10811 was deposited at the DeutscheSammlung von Mikroorganismen und Zellkulturen on Jun. 30, 1995 accordingto the Budapest Treaty. The plasmid was digested with the restrictionendonucleases BamHI and NotI The 4153 bp vector part and the 1211 bpBamHI-NotI fragment were isolated. Equal portions of the 1211 bpfragment were digested with respectively HgiAI and EcoRV and the 487 bpBamHI-HgiAI and 690 bp EcoRV-NotI fragments were isolated.

[0362] These fragments and the vector part were ligated in the presenceof 5 fold molar excess of a synthetic DNA fragment, resulting from theannealing of two single stranded DNA oligomers: 18802:CACTGGCGGCGACCTGGGATCTAACCACTTCGAT 18803:ATCGAAGTGGTTAGATCCCAGGTCGCCGCCTGTGCTC

[0363] The ligation mixture was transformed into E. coli strain XL1, andfrom the resulting transformants Thielavia terrestris/Q119H was isolatedand verified by DNA sequencing.

[0364] All the cellulase variants ware produced by cloning the gene andtransforming the gene into Aspergillus oryzae using a plasmid with thegene inserted between the fungal amylase promoter and the AMG terminatorfrom A. niger. [Christensen, T. Wöldike, H. Boel, E., Mortensen, S. B.,Hjortsoj, K., Thim, L. and Hansen, M. T. (1988) Biotechnology 6:1419-1422].

[0365] The cellulases with a cellulose binding domain CBD were purifiedby exploiting their binding to Avicel. The cloned product was recoveredafter fermentation by separation of the extracellular fluid from theproduction organism. The cellulase was then highly purified by affinitychromatography using 150 gram of Avicel in a slurry with 20 mMsodiumphosphate pH 7.5. The Avicel slurry was mixed with the crudefermentation broth which in total contains about 1 gram of protein.After mixing at 4° C. for 20 min, the Avicel-bound enzyme is packed intoa column with a dimension of 50 times 200 mm about 400 ml total.

[0366] The column is washed with the 200 ml buffer, then washed with 0.5M NaCl in the same buffer until no more protein elutes, and washed with500 ml buffer (20 mM Tris pH 8.5). Finally the pure full length enzymeis eluted with 1% Triethylamine pH 11.8. The eluted enzyme solution isadjusted to pH 8 and concentrated using a Amicon cell unit with amembrane DOW GR61PP (polypropylene with a cut off of 20 KD) to 5 mgprotein per ml. The enzymes have all been purified yielding a singleband on SDS-PAGE.

[0367] Cellulases which natural lack CBD or the linker has beenproteolytic cleaved or in which the CBD has been removed by introducinga stop codon after the catalytic domain, can not be purified usingAvicel. The extracellular proteins are recovered free from theproduction organism. The core cellulases were purified free ofAspergillus proteins by cation exchange chromatography. The fermentationbroth was adjusted to pH 3.5 and filtered to remove the precipitatingproteins. Then the proteins were ultra filtrated (concentrated andwashed with water) on a DOW GR81PP membrane with a cut off 6 KD untilthe conductivity of the eluate is below 1000 mS/cm. The sample wasfinally applied to a S-Sepharose column equilibrated with a 20 mMcitrate buffer pH 3.5 .

[0368] The enzyme will bind to the S-Sepharose at this low pH and it iseluted as a single peak using a NaCl gradient from 0 to 500 mM. Theeluted pure enzyme was concentrated on a Amicon cell with the DOW GR81PPmembrane. All purified cellulases gave a single band in SDS-PAGE.

[0369] The specific activity data are summarized in the following table:Specific activity Enzyme/variant [%] Humicola insolens 100  Thielaviaterrestris 35 Thielavia terrestris/Q119H 92

[0370] From this experiment it is seen that by introducing the mutationQ119H into the Thielavia terrestris cellulase, the specific activity ifthe resulting cellulase variants was increased to the level of that ofthe homologous Humicola insolens cellulase.

Example 2

[0371]Thielavia terrestris Variant with Improved Alkaline PerformanceProfile

[0372] In this experiment the Thielavia terrestris/Q119D was constructedas described in example 1 but using the following construction: For easycassette swap and standard primer utilization, the CT1 encoding DNA wasfurnished with a C-terminal Xba1 site and subcloned into the pCaHj418vector as described below. PCT1 was used as template in a Pwo polymerasePCR, 94 C2′ −3x(94 C,30″-72 C,1′)-25x(94 C,30″-55 C, 30″-72, C 1′) −72C,5′ applying the two primers 8939: CGACTTCAATGTCCAGTCGG 25335:GCGCTCTAGAGGATTAAAGGCACTGC

[0373] The resulting 718 bp PCR product was digested with Sal1 and Xba1and the 165 bp fragment was isolated. This fragment was ligated togetherwith the 833 bp BamH1-Sal1 fragment from pCT1-2 into the 4.1 kbXba1-BamH1 vector fragment of pCaHj418.

[0374] From this ligation pCT1418 was isoltated from E. colitransformants.

[0375] PCT2 was constructed by the Chameleon™ Double-stranded,site-directed Mutagenesis kit (from Stratagene) as described above withpCT1418 as template, the S/M primer as selection primer and thefollowing mutagenic primer: 109330: CGACCTGGGATCGAACGACTTCGATATCGCCATGC

[0376] A successfully mutated plasmid pCT2 was isolated, verified by DNAsequencing and transformed into Aspergillus oryzae strain JaL228.

[0377] The Thielavia terrestris cellulase and the Thielaviaterrestris/Q119D variant was tested for activity towards PASC asdescribed in example 9 at pH 7.0 and pH 10.0.

[0378] The results are presented in the table below which shows theactivity at pH10 compared to the activity at pH 7. This demonstratesthat the Thielavia terrestris/Q119D variant has relatively more alkalineactivity as compared to the parent Thielavia terrestris. Relativeactivity pH 10/pH 7 [%] Thielavia terrestris 27 Thielaviaterrestris/Q119D 62

Example 3

[0379] Construction of a Cellulase Hybrid Variant

[0380] The plasmid pCT3 embodies DNA encoding the Thielavia terrestrisendoglucanase core enzyme and followed by the linker CBD of Humicolagrisea.

[0381] pCT3 was constructed by means of sequence overlap extension PCR,applying PWO polymerase.

[0382] From a cDNA clone of Humicola grisea a 415 bp fragment wasgenerated by the following primers: 109452:CGACTCCAGCTTCCCCGTCTTCACGCCCCC 107819:CGAGCTTCTAGATCTCGACTAGAGGCACTGGGAG

[0383] From pCT1418 (disclosed in example 2) a 876 bp PCR fragment wasgenerated by the following primers: 101621: GGATGCCATGCTTGGAGGATAGCAACC107823: GGGGGCGTGAAGACGGGAAGCTGGAGTCG

[0384] For both reactions the following set up was used: 96_C, 140-3x(94_C, 30″-50_C, 1′-72_C, 1′)-25x(94_C, 30″-61_C, 30″-72_C, 1′)-72_C,7′.

[0385] The isolated PCR fragments were applied as template in anassembly PCR reaction with primers 101621 and 107819: 94_C, 1′-3x(94_C,30″-70_C, 1′-72_C, 2′)-20x(94_C, 30″-61_C, 30″-72_C, 1,5″)-72_C, 7′. Theresulting 1261 bp PCR product was isolated cut by restriction enzymesBamH1 and Xba1 and the resulting 1172 bp DNA fragment was isolated andligated into the 4.1 kb vector fragment of BamH1-Xba1 digested pCaHj418.

[0386] Correct clones were isolated and verified by DNA sequencing ofplasmids isolated from E. coli XL1 transformants resulting aboveligation reaction. cDNA sequence of Huraicola grisea:CAAGAACCTCACACTCATTTTATTCACGCTCATTTATTCTAAAACTTCAATATGCGCTCTGCTCCTATTTTCCGCACGGCCCTGGCGGCTGCGCTCCCCCTTGCCGCACTCGCCGCCGATGGCAAGTCGACCAGATACTGGGACTGCTGCAAGCCATCGTGCTCTTGGCCCGGAAAGGCACTCGTGAACCAGCCTGTCTTCACTTGCGACGCCAAATTCCAGCGCATCACCGACCCCAATACCAAGTCGGGCTGCGATGGCGGCTCGGCCTTTTCGTGTGCTGACCAGACCCCCTGGGCTCTGAACGACGATGTCGCCTATGGCTTCGCTGCCACGGCTATTTCGGGTGGATCGGAAGCCTCGTGGTGCTGCGCATGCTACGCTCTTACTTTCACCTCGGGCCCTGTGGCCGGCAAGACCATGGTCGTCCAGTCGACCAACACCGGCGGCGATCTCGGCAGCAACCATTTCGACCTCCAGATTCCAGGCGGCGGTGTCGGCATCTTTGATGGGTGCACCCCCCAGTTCGGAGGTCTCGCTGGCGAACGCTACGGTGGCATCTCAGACCGCAGCTCCTGCGACTCGTTCCCTGCGGCGCTCAAGCCCGGCTGCCTCTGGCGCTTCGATTGGTTCAAGAACGCCGACAACCCGACCTTTACCTTCAAGCAGGTGCAGTGCCCCGCCGAGCTTGTTGCCAGGACCGGCTGCAAGCGCGAGGATGACGGCAACTTCCCCGTCTTCACGCCCCCCGCGGGTAGCAACACCGGCGGTAGCCAGTCGAGCTCCACTATCGCTTCCAGCTCGACCTCCAAGGCTCAGACTTCGGCCGCCAGCTCCACCTCCAAGGCTGTCGTGACTCCCGTCTCCAGCTCCACCTCGAAGGCCGCTGAGGTCCCCAAATCCAGCTCGACCTCCAAGGCTGCCGAGGTCGCCAAGCCCAGCTCAACTTCGACCTCGACCTCGACCTCGACCAAGGTCAGCTGCTCTGCGACCGGTGGCTCCTGCGTCGCTCAGAAGTGGGCGCAGTGCGGCGGCAATGGCTTCACCGGCTGCACGTCGTGCGTCAGCGGCACCACCTGCCAGAAGCAAAATGACTGGTACTCCCAGTGCCTCTAAGTCGTTTGTAGTAGCAGTTTGAAGGATGTCAGGGATGAGGGAGGGAGGAGTGGGGGAAAAGTACGCCGCAGTTTTTTGGTAGACTTACTGTATTGTTGAGTAATTACCCATTCGCTTCTTGTACGAAAAAAAAA AAAAAAAAAA

Example 4

[0387] Construction of Variants of a Hybrid Cellulase

[0388] The plasmid pPsF45 embodies DNA encoding the Pseudomonascellolytica endoglucanase core enzyme headed by the H. insolens EGVendoglucanase signal peptide and followed by the linker CBD of sameenzyme.

[0389] Two variants of this hybrid enzyme were constructed by means ofthe above-mentioned Stratagene Chameleon® kit: PsF45/H15S, andPsF45/Q119H (cellulase numbering) by application of the followingmutagenic primers PsF45/H15S: GCTGCAAGCCGTCCTGTGGCTGGAGCGCTAACGTGCCCGCGPsF45/Q119H: CGATGTTTCCGGAGGCCACTTTGACATTCTGGTTCC

[0390] Deviations from template sequence are indicated in bold type.

[0391] The selection primer was converting the unike Sca1 site in thelactamase gene of the plasmid to a Mlu1 site:

[0392] GAATGACTTGGTTGACGCGTCACCAGTCAC

[0393] The two variants were verified by DNA sequencing and one correctversion of each variant was identified.

[0394] The two plasmids emharboring the variant sequences pPsF45H15S andpPsF45Q119H were used to transform A. oryzae strain JaL142 together withthe AMDS selection plasmid pToC202. From the resulting transformantsLaC2829 and LaC 2830 were isolated after 3 reisolation steps via spores.

Example 5

[0395] Removal of Disulfide Bridges

[0396] Disulfide bridges are known to stabilize protein structures. Theremoval of disulfide bridges in a cellulase will destabilizes the enzyme(thermostability) while retaining significant activity. This can beuseful in applications where a fast inactivation of the enzyme ispreferred, e.g. in denim or textile applications or for low temperatureprocesses.

[0397] In this example Humicola insolens EGV cellulase and five variantsof Humicola insolens cellulase were constructed mutating either one orboth residues involved in a disulfide bridge. The specific activity wasmeasured as disclosed under Materials and Methods. The meltingtemperature of the enzymes were measured using Differential ScanningCalometry, DSC. DSC was done at neutral pH (7.0) using a MicroCalc Inc.MC calorimeter with a constant scan rate and raising the temperaturefrom 20° C. to 90° C. at a rate of 90° C. per hour.

[0398] The results are presented in the table below which shows thatremoval of a disulfide bridge leads to a variant with a significantlylower melting temperature but retaining significant activity. Specificactivity Melting temp. [%] [° C.] Humicola insolens 100  81 Humicolainsolens/C12G, C47M 15 63.7 Humicola insolens/C12M, C47G 53 64.3Humicola insolens/C47G 48 57.3 Humicola insolens/C87M, C199G 75 63.4Humicola insolens/C16M, C86G 103  59.2

Example 6

[0399] Mutation of Conserved Residues in the Binding Cleft <5 Å fromSubstrate

[0400] When comparing the positions within a distance of 5 Å from thesubstrate to the sequence alignment in Table 1 the type of amino acidresidue at these positions are conserved in the aligned cellulases forthe following positions: 6, 7, 8, 9, 10, 11, 12, 18, 45, 112, 114, 121,127, 128, 130, 132, 147, 148, 149. Conserved residues are normallythought to be extremely important for the activity, but the inventorshave found that a certain variability is allowed while maintainingsignificant activity. Only the two residues D10 and D121 (cellulasenumbering) are necessary to maintain reasonable activity.

[0401] Variants of the Humicola insolens EGV cellulase were prepared andthe specific activity was measured as disclosed in Materials andMethods.

[0402] The type of mutations and the variants specific activity aresummarized in the following table: Specific activity Variant [%]Humicola insolens 100  Humicola insolens/T6S 34 Humicola insolens/R7I 33Humicola insolens/R7W 29 Humicola insolens/Y8F 67 Humicola insolens/W9F83 Humicola insolens/C12M, C47G 53 Humicola insolens/W18Y 49 Humicolainsolens/W18F 53 Humicola insolens/S45T 85 Humicola insolens/S45N 85Humicola insolens/D114N 6 Humicola insolens/F132D 11 Humicolainsolens/Y147D 34 Humicola insolens/Y147C 30 Humicola insolens/Y147W 74Humicola insolens/Y147V 33 Humicola insolens/Y147R 45 Humicolainsolens/Y147G 34 Humicola insolens/Y147Q 41 Humicola insolens/Y147N 53Humicola insolens/Y147K 45 Humicola insolens/Y147H 75 Humicolainsolens/Y147P 57 Humicola insolens/Y147S 55

[0403] From this experiment it is seen that mutating conserved residuesin the binding cleft can be performed while retaining significantactivity of the cellulase variant.

Example 7

[0404] Mutation of Non-Conserved Residues in the Binding Cleft <5 Å fromthe Substrate

[0405] Based on the sequence alignment in Table 1 and the disclosedcomputer modeling method the following residues located within adistance of 5Å from the substrate and not being conserved amongst thealigned sequences in were identified as points of interest for makingcellulase variants.

[0406] In this experiment non-conserved residues located no more than 5Å from the substrate were modified in the Humicola insolens EGVcellulase and the specific activity was measured as described underMaterials and Methods.

[0407] The type of mutations and the variants specific activity aresummarized in the following table: Specific activity [%] Humicolainsolens 100 Humicola insolens/R4H 73 Humicola insolens/R4Q 70 Humicolainsolens/K13L 37 Humicola insolens/K13R 100 Humicola insolens/K13Q 38Humicola insolens/P14A 99 Humicola insolens/P14T 71 Humicolainsolens/S15T 18 Humicola insolens/S15H 10 Humicola insolens/C16M, C86G103 Humicola insolens/A19P 51 Humicola insolens/A19T 84 Humicolainsolens/A19G 78 Humicola insolens/A19S 89 Humicola insolens/K20G 91Humicola insolens/D42Y 102 Humicola insolens/D42W 103 Humicolainsolens/C47G 48 Humicola insolens/E48D 93 Humicola insolens/E48Q 71Humicola insolens/E48D, P49* 88 Humicola insolens/E48N, P49* 79 Humicolainsolens/S110N 94 Humicola insolens/L115I 18 Humicola insolens/G116D 71Humicola insolens/H119R 15 Humicola insolens/H119Q 39 Humicolainsolens/H119F 11 Humicola insolens/N123A 61 Humicola insolens/N123M 80Humicola insolens/N123Q 76 Humicola insolens/N123Y 8 Humicolainsolens/N123D 86 Humicola insolens/V129L 72 Humicola insolens/D133N 102Humicola insolens/D178N 81

[0408] From this experiment it is seen that most of the non-conservedresidues in the binding cleft can be mutated while retaining all or mostof the activity of the cellulase.

Example 8

[0409] Resistance to Anionic Surfactants in Detergent

[0410] A. Variants of the present invention may show improvedperformance with respect to an altered sensitivity towards anionictensides. Anionic tensides are products frequently incorporated intodetergent compositions. Unfolding of cellulases tested so far, isaccompanied by a decay in the intrinsic fluorescence of the proteins.The intrinsic fluorescence derives from Trp side chains (and to asmaller extent Tyr side chains) and is sensitive to the hydrophobicityof the side chain environment. Unfolding leads to a more hydrophilicenvironment as the side-chains become more exposed to solvent, and thisquenches fluorescence.

[0411] Fluorescence is followed on a Perkin/Elmer™ LS50 luminescencespectrometer. In practice, the greatest change in fluorescence onunfolding is obtained by excitation at 280 nm and emission at 345 nm.Slit widths (which regulate the magnitude of the signal) are usually 5nm for both emission and excitation at a protein concentration of 5μg/ml. Fluorescence is measured in 2-ml quartz cuvettes thermostattedwith a circulating water bath and stirred with a small magnet. Themagnet-stirrer is built into the spectrometer.

[0412] Unfolding can be followed in real time using the availablesoftware. Rapid unfolding (going to completion within less than 5-10minutes) is monitored in the TimeDrive option, in which the fluorescenceis measured every few (2-5) seconds. For slower unfolding, four cuvettescan be measured at a time in the cuvette-holder using the WavelengthProgram option, in which the fluorescence of each cuvette is measuredevery 30 seconds. In all cases, unfolding is initiated by adding a smallvolume (typically 50 μl) of concentrated enzyme solution to thethermostatted cuvette solution where mixing is complete within a fewseconds due to the rapid rotation of the magnet.

[0413] Data are measured in the software program GraphPad Prism.Unfolding fits in all cases to a single-exponential function from whicha single half-time of unfolding (or unfolding rate constant) can beobtained.

[0414] Typical unfolding conditions are:

[0415] a. 10 mM CAPS pH 10, 1000 ppm LAS, 40° C.

[0416] b. 10 mM HEPES pH 10, 200 ppm LAS, 25° C.

[0417] In both cases, the protein concentration is 5-10 μg/ml (theprotein concentration is not crucial, as LAS is in excess). Under theseconditions, the unfolding of Humicola insolens cellulase can be comparedwith other enzymes (Table 1). This enables us to draw up the followingranking order for stability against anionic tenside:

[0418]Thielavia terrestris/Q119H≅Thielavia terrestris>>Humicolainsolens≅Humicola insolens/H119Q. t½ pH 10 (s) t½ pH 7 (s) Cellulase(1000 ppm LAS, 40° C.) (200 ppm LAS, 25° C.) Humicola insolens 48  28Humicola insolens/ 63  9a H119Q Thielavia terrestris 970  690 Thielaviaterrestris/ 1100  550 Q1191H

[0419] a Unfolding is double-exponential. The t½ of the slower phase isapprox. 120 sec.

[0420] B. The alteration of the surface electrostatics of an enzyme willinfluence the sensibility towards anionic tensides such as LAS (linearalkylbenzenesulfonate). Especially variants where positive chargedresidues have been removed and/or negatively charged residues have beenintroduced will increase the resistance towards LAS, whereas theopposite, i.e. the introduction of positively charged residues and/orthe removal of negatively charged residues will lower the resistancetowards LAS. The residues Arg (R), Lys (K) and His (H) are viewed aspositively or potentially positively charged residue and the residuesAsp (D), Glu (E) and Cys (C) if not included in a disulphide bridge areviewed as negatively or potentially negatively charged residues.Positions already containing one of these residues are the primarytarget for mutagenesis, secondary targets are positions which has one ofthese residues on an equivalent position in another cellulase, and thirdtarget are any surface exposed residue. In this experiment wild typeHumicola insolens cellulase are being compared to Humicola insolenscellulase variants belonging to all three of the above groups, comparingthe stability towards LAS in detergent.

[0421] Cellulase resistance to anionic surfactants was measured asactivity on PASC (phosphoric acid swollen cellulose) in the presence ofanionic surfactant vs. activity on PASC in the absence of anionicsurfactant.

[0422] The reaction medium contained 5.0 g/l of a commercial regularpowder detergent from the detergent manufacturer NOPA Denmark. Thedetergent was formulated without surfactants for this experiment and pHadjusted to pH 7.0. Further the reaction medium included 0.5 g/l PASCand was with or without 1 g/l LAS (linear alkylbenzenesulphonate), whichis an anionic surfactant, and the reaction proceeded at the temperature30° C. for 30 minutes. Cellulase was dosed at 0.20 S-CEVU/l. After the30 minutes of incubation the reaction was stopped with 2 N NaOH and theamount of reducing sugar ends determined through reduction ofp-hydroxybenzoic acid hydrazide. The decrease in absorption of reducedp-hydroxybenzoic acid hydrazide relates to the cellulase activity.

[0423] The type of mutation and the resistance towards LAS for variantswith increased LAS resistance is summarized in the following table:Relative LAS resistance Variant [%] Humicola insolens 100 Humicolainsolens/R158E 341 Humicola insolens/Y8F, W62E, A162P, 179 Humicolainsolens/R158E, A162P 347 Humicola insolens/R158G 322 Humicolainsolens/S152D 161 Humicola insolens/R158E/R196E 319 Humicolainsolens/R158E, D161P, A162P 351 Humicola insolens/R4H, R158E, D161P,A162P 344 Humicola insolens/H119Q 148 Humicola insolens/Y8F, W62E,R252L, Y280F 131 Humicola insolens/R252L, Y280F 133 Humicolainsolens/W62E, A162P 130 Humicola insolens/W62E, A162P 129 Humicolainsolens/S117D 143 Humicola insolens/A57C, A162C 134 Humicolainsolens/N154D 149 Humicola insolens/R4H, D161P, A162P, R196E 134

[0424] From this table it is seen that mutations of residues resultingin the removal of positively charged residue and/or the introduction ofa negatively charged residue increase the resistance towards LAS.

[0425] As described above the type of mutation and the resistancetowards LAS for variants with decreased LAS resistance is summarized inthe following table: Relative LAS resistance Variant [%] Humicolainsolens 100 Humicola insolens/Y147H 71 Humicola insolens/E192P 52Humicola insolens/D161P, A162P 64 Humicola insolens/D67T 44 Humicolainsolens/Q36T, D67T 67 Humicola insolens/D66N 47 Humicola insolens/D67N71 Humicola insolens/V64R 58 Humicola insolens/N65R 48 Humicolainsolens/T93R 60 Humicola insolens/Q36T, D67T, A83T 64 Humicolainsolens/E91Q 71 Humicola insolens/A191K 63 Humicola insolens/D42W 67Humicola insolens/S117K 62 Humicola insolens/R4H, A63R, N65R, D67R 54Humicola insolens/D133N 0 Humicola insolens/D58A 15 Humicolainsolens/D67R 39 Humicola insolens/A63R 38 Humicola insolens/R37N, D58A6 Humicola insolens/K175R 32 Humicola insolens/D2N 43 Humicolainsolens/N65R, D67R 40 Humicola insolens/T136D, G141R 5 Humicolainsolens/Y147K 17 Humicola insolens/Y147R 1 Humicola insolens/D161P 35Humicola insolens/D66P 40 Humicola insolens/D66A, D67T 39 Humicolainsolens/DG7T, *143NGT 7 Humicola insolens/Q36T, D67T, *143NGT 0Humicola insolens/N65R, D67R, S76K 22 Humicola insolens/W62R 25 Humicolainsolens/S117R, F120S 31 Humicola insolens/K13R 16 Humicolainsolens/D10E 0

[0426] From this table it is seen that mutations of residues resultingin the introduction of positively charged residue and/or the removal ofa negatively charged residue decrease the resistance towards LAS.

Example 9

[0427] Alteration of pH Activity Profile

[0428] The pH activity profile of a cellulase is governed by the pHdependent behavior of specific titratable groups, typically the acidicresidues in the active site. The pH profile can be altered by changingthe electrostatic environment of these residues, either by substitutionof residues involving charged or potentially charged groups such as Arg(R), Lys (K), Tyr (Y), His (H), Glu (E), Asp (D) or Cys (C) if notinvolved in a disulphide bridge or by changes in the surfaceaccessibility of these specific titratable groups by mutations in thebiding cleft within 5 Å of the substrate.

[0429] In this example Humicola insolens cellulase and variants ofHumicola insolens cellulase involving substitution of charged orpotentially charged residues have been tested for activity towards PASCat pH 7 and pH 10, respectively.

[0430] In order to determine the pH optimum for cellulases we haveselected organic buffers because it is common known that e.g. borateforms covalent complexes with mono- and oligo-saccharides and phosphatecan precipitate with Ca-ions. In DATA FOR BIOCHEMICAL RESEARCH ThirdEdition OXFORD SCIENCE PUBLICATIONS page 223 to 241, suitable organicbuffers has been found. In respect of their pKa values we decided to useNa-acetate in the range 4-5.5, MES at 6.0, MOPS in the range 6.5-7.5,Na-barbiturate 8.0-8.5 and glycine in the range 9.0-10.5.

[0431] Method:

[0432] The method is enzymatic degradation of carboxy-methyl-cellulose,at different pH's. Buffers are prepared in the range 4.0 to 10.5 withintervals of 0.5 pH unit. The analyze is based on formation of newreducing ends in carboxy-methyl-cellulose, these are visualized byreaction with PHBAH in strong alkaline environment, were they forms ayellow compound with absorption maximum at 410 nm.

[0433] Experimental Protocol:

[0434] Buffer preparation: 0.2 mol of each buffer substance is weighedout and dissolved in 1 liter of Milli Q water. 250 ml 0.2M buffersolution and 200 ml Milli Q water is mixed. The pH are measured usingRadiometer PHM92 labmeter calibrated using standard buffer solutionsfrom Radiometer. The pH of the buffers are adjusted to actual pH using4M NaOH or 4M HCl and adjusted to total 500 ml with water. Whenadjusting Na-barbiturate to pH 8.0 there might be some precipitation,this can be re-dissolved by heating to 50° C.

[0435] Acetic acid 100% 0.2 mol=12.01 g.

[0436] MES 0.2 mol=39.04 g.

[0437] MOPS 0.2 mol=41.86 g.

[0438] Na-barbiturate 0.2 mol=41.24 g.

[0439] Glycine 0.2mol=15.01 g.

[0440] Buffers:

[0441] pH: 4.0, 4.5, 5.0 & 5.5 Na-acetate 0.1M

[0442] pH: 6.0 Na-MES 0.1M

[0443] pH: 6.5, 7.0 & 7.5 Na-MOPS 0.1M

[0444] pH: 8.0 & 8.5 Na-barbiturate 0.1M

[0445] pH: 9.0, 9.5, 10.0 & 10.5 Na.glycine 0.1M

[0446] The actual pH is measured in a series treated as the main values,but without stop reagent, pH is measured after 20 min. incubation at 40°C.

[0447] Substrate Preparation:

[0448] 2.0 g CMC, in 250 ml conic glass flask with a magnet rod, ismoistened with 2.5 ml. 96% ethanol, 100 ml. Milli Q water is added andthen boiled to transparency on a heating magnetic stirrer. Approximately2 min. boiling. Cooled to room temperature on magnetic stirrer.

[0449] Stop Reagent:

[0450] 1.5 g PHBAH and 5 g K-Na-tartrate dissolved in 2% NaOH.

[0451] Procedure:

[0452] There are made 3 main values and 2 blank value using 5 ml glasstest tubes. (1 main value for pH determination) Main values Blank valueBuffer 1.0 ml. 1.0 ml. Substrate CMC 0.75 ml. 0.75 ml. Mix 5 sec. 5 sec.Preheat 10 min./40° C. — Enzyme 0.25 ml. — Mix 5 sec. — Incubation 20min./40° C. room temp. PHBAH-reagent 1 ml. 1 ml. Mix 5 sec. — Enzyme —0.25 ml. Mix — 5 sec.

[0453] Mixing on a Heidolph REAX 2000 mixer with permanent mix andmaximum speed (9). No stirring during incubation on water bath withtemperature control. Immediately after adding PHBAH-reagent and mixingthe samples are boiled 10 min. Cooled in cold tap water for 5 min.Absorbance read at 410 nm.

[0454] Determination of Activity

[0455] The absorbance at 410 nm from the 2 Main values are added anddivided by 2 and the 2 Blank values are added and divided by 2, the 2mean values are subtracted. The percentages are calculated by using thehighest value as 100%.

[0456] The measured pH is plotted against the relative activity.

[0457] Buffer Reagents:

[0458] Acetic acid 100% from MERCK cat.no.1.00063, batcno.K20928263 422,pKa 4.76, MW 60.05;

[0459] MES (2[N-Morpholino]ethanesulfonic Acid) from SIGMA cat.no.M-8250, batchno. 68F-5625, pKa 6.09, MW 195.2;

[0460] MOPS (3-[N-Morpholino]propanesulfonic Acid) from SIGMA cat.no.M-1254, batchno. 115F-5629, pKa 7.15, MW 209.3;

[0461] Na-barbiturate (5,5-Diethylbarbituric acid sodium salt) fromMERCK cat.no. 6318, batch no. K20238018 404, pKa 7.98, MW 206.2;

[0462] Glycine from MERCK cat.no.4201, batch no. K205535601 405, pKa9.78, MW 75.07;

[0463] PHBAH (p-HYDROXY BENZOIC ACID HYDRAZIDE): From SIGMAcat.no.H-9882, batch no. 53H7704;

[0464] K-Na-tartrate (Potassium sodium tartrate tetrahydrate) from MERCKcat.no. 8087, batch no. A653387 304;

[0465] NaOH (Sodium hydroxyde) from MERCK cat.no. 1.06498, batch no.C294798 404;

[0466] CMC (Carboxy Methyl Cellulose) supplied by Hercules (FMC)7LF(November 1989).

[0467] Cellulase resistance to anionic surfactants was measured asactivity on PASC (phosphoric acid swollen cellulose) at neutral pH (pH7.0) vs. activity on PASC at alkaline pH (pH 10.0). The reaction mediumcontained 5.0 g/l of a commercial regular powder detergent from thedetergent manufacturer NOPA Denmark. The pH was adjusted to pH 7.0 andpH 10.0, respectively. Further the reaction medium included 0.5 g/lPASC, and the reaction proceeded at the temperature 30° C. for 30minutes. Cellulase was dosed at 0.20 S-CEVU/l. After the 30 minutes ofincubation the reaction was stopped with 2 N NaOH and the amount ofreducing sugar ends determined through reduction of p-hydroxybenzoicacid hydrazide. The decrease in absorption of reduced p-hydroxybenzoicacid hydrazide relates to the cellulase activity.

[0468] Results:

[0469] The results are presented in the table below, the activity atpH10 relative to pH7 is compared to that of wild type Humicola insolenscellulase. PASC activity pH10/pH7 relative to wild type Variant [%]Humicola insolens 100 Humicola insolens/S76K, S117D 120 Humicolainsolens/V129L 133 Humicola insolens/R4H, A63R, N65R, D67R 120 Humicolainsolens/R252L, Y280F 115 Humicola insolens/D161P, A162P 117 Humicolainsolens/A57C, A162C 110 Humicola insolens/S76K 117 Humicolainsolens/D161P, A162P, R196E 113 Humicola insolens/Q36T, D67T, A83T 111Humicola insolens/W62R 112 Humicola insolens/D42Y 110 Humicolainsolens/S76K, A78K 114 Humicola insolens/S76K, A78R 118

[0470] From the above table it is seen that the relative alkalineactivity can be increased by creating variants involving potentiallycharged residues and/or by altering residues in the binding cleft lessthat 5 Å from the substrate.

[0471] Similarly the following table shows that the relative acidicactivity can be increased by other mutations involving potentiallycharged residues and/or by altering residues in the binding cleft lessthan 5 Å from the substrate. PASC activity pH10/pH7 relative to wildtype Variant [%] Humicola insolens 100  Humicola insolens/D58A 83Humicola insolens/Y280W 90 Humicola insolens/D67R 89 Humicolainsolens/A63R 85 Humicola insolens/Y8F 82 Humicola insolens/W62E 82Humicola insolens/R37N, D58A 84 Humicola insolens/K175G 81 Humicolainsolens/K175R 82 Humicola insolens/Y8F, M104Q 83 Humicola insolens/Y8F,W62E, R252L, Y280F 83 Humicola insolens/W62E, A162P 87 Humicolainsolens/Y8F, W62E, A162P, 88 Humicola insolens/Y147H 90 Humicolainsolens/Y147N 90 Humicola insolens/Y147Q 85 Humicola insolens/Y147W 85Humicola insolens/E192P 89 Humicola insolens/R158G 83 Humicolainsolens/S152D 90 Humicola insolens/K13Q 82 Humicola insolens/R37P 82Humicola insolens/S45T 87 Humicola insolens/E48D 86 Humicolainsolens/R7I 83 Humicola insolens/P14A 84 Humicola insolens/A19G 90Humicola insolens/A19T 90 Humicola insolens/R4H, D161P, A162P, R196E 88Humicola insolens/D133N 80 Humicola insolens/D40N 40 Humicolainsolens/Y90F 72 Humicola insolens/A63D 78 Humicola insolens/ 25 G127S,I131A, A162P, Y280F, R252L Humicola insolens/Y147S 39 Humicolainsolens/Y147F 71 Humicola insolens/T6S 44 Humicola insolens/S55E 14Humicola insolens/N123D 35 Humicola insolens/N123Y 71 Humicolainsolens/R158E 78 Humicola insolens/T136D, G141R 57 Humicolainsolens/G127S, I131A, A162P 52 Humicola insolens/ 35 W62E, G127S,I131A, A162P, Y280F, R252L Humicola insolens/W62E, G127S, I131A, A162P58 Humicola insolens/W62E, G127S, I131A 64 Humicola insolens/ 80 W62E,G127S, I131A, Y280F, R252L Humicola insolens/H119Q 57 Humicolainsolens/Y8F, W62E 61 Humicola insolens/W62E, A162P 76 Humicolainsolens/W62E, A162P 80 Humicola insolens/R158E, A162P 80 Humicolainsolens/Y8F, Y147S 63 Humicola insolens/Y147R 54 Humicolainsolens/Y147V 22 Humicola insolens/Y147C 67 Humicola insolens/Y147D 60Humicola insolens/N154D 74 Humicola insolens/R158E, R196E 79 Humicolainsolens/R158E, D161P, A162P 70 Humicola insolens/D67T, *143NGT 65Humicola insolens/Q36T, D67T, *143NGT 53 Humicola insolens/143*NGW,Q145D 53 Humicola insolens/L142P, 143*NGW, Q145E 42 Humicolainsolens/N65R, D67R, S76K 60 Humicola insolens/A63R, N65R, D67R 77Humicola insolens/T93R 80 Humicola insolens/S76R 70 Humicolainsolens/S117R, F120S 58 Humicola insolens/N123Q 63 Humicolainsolens/N123M 49 Humicola insolens/N123A 80 Humicola insolens/E48D,P49* 66 Humicola insolens/S55Y 61 Humicola insolens/S55M 48 Humicolainsolens/W18F 54 Humicola insolens/S45N 71 Humicola insolens/R7W 58Humicola insolens/K13R 72 Humicola insolens/R7L 74 Humicolainsolens/S15T 38 Humicola insolens/W18Y 37 Humicola insolens/C16M, C86G67 Humicola insolens/K13L 59 Humicola insolens/C12M, C470 12 Humicolainsolens/W9F 62 Humicola insolens/C47G 58 Humicola insolens/C12G, C47M 0 Humicola insolens/D10E  0 Humicola insolens/R7K 49

[0472] Accordingly, this example demonstrates that the relative activitypH profile can be altered towards acidic or alkaline pH by creation ofvariants involving potentially charged residues and/or by alteringresidues in the binding cleft less that 5 Å from the substrate.

Example 10

[0473] Wash Performance of Cellulases made Resistant to AnionicSurfactants

[0474] Application effect of a cellulase made resistant to anionicsurfactants vs. application effect of the native cellulase was measuredas ‘color clarification’ of worn black cotton swatches laundered withcellulase in a 0.1 liter mini-Terg-o-Meter. Laundering was done invarying concentrations of anionic surfactant.

[0475] The reaction medium contained phosphate buffer pH 7.0 and varyingconcentrations of LAS in the range 0.2-1.0 g/L. Two swatches werelaundered at 40° C. for 30 minutes, rinsed and then dried. Thislaundering cycle was repeated four times. All enzymes was tested at eachLAS concentration.

[0476] Finally the black cotton swatches were graded against a standardof similar swatches washed with varying dosages of the native cellulase,the fungal ~43 kD endo-β-1,4-glucanase from Humicola insolens, DSM 1800,(commercially available under the tradename Carezyme®), and the effectexpressed in PSU (panel score units). LAS concentration 0.2 0.4 0.6 0.81.0 Variant g/l g/l g/l g/l g/l Humicola insolens 15 0 0 0 0 Humicolainsolens/R158E 30 14 30 22 11 Humicola insolens/R158G 20 18 20 33 28

[0477]

1 26 1 202 PRT Cellulase variants 1 Ala Asp Gly Arg Ser Thr Arg Tyr TrpAsp Cys Cys Lys Pro Ser Cys 1 5 10 15 Gly Trp Ala Lys Lys Ala Pro ValAsn Gln Pro Val Phe Ser Cys Asn 20 25 30 Ala Asn Phe Gln Arg Ile Thr AspPhe Asp Ala Lys Ser Gly Cys Glu 35 40 45 Pro Gly Gly Val Ala Tyr Ser CysAla Asp Gln Thr Pro Trp Ala Val 50 55 60 Asn Asp Asp Phe Ala Leu Gly PheAla Ala Thr Ser Ile Ala Gly Ser 65 70 75 80 Asn Glu Ala Gly Trp Cys CysAla Cys Tyr Glu Leu Thr Phe Thr Ser 85 90 95 Gly Pro Val Ala Gly Lys LysMet Val Val Gln Ser Thr Ser Thr Gly 100 105 110 Gly Asp Leu Gly Ser AsnHis Phe Asp Leu Asn Ile Pro Gly Gly Gly 115 120 125 Val Gly Ile Phe AspGly Cys Thr Pro Gln Phe Gly Gly Leu Pro Gly 130 135 140 Gln Arg Tyr GlyGly Ile Ser Ser Arg Asn Glu Cys Asp Arg Phe Pro 145 150 155 160 Asp AlaLeu Lys Pro Gly Cys Tyr Trp Arg Phe Asp Trp Phe Lys Asn 165 170 175 AlaAsp Asn Pro Ser Phe Ser Phe Arg Gln Val Gln Cys Pro Ala Glu 180 185 190Leu Val Ala Arg Thr Gly Cys Arg Arg Ala 195 200 2 202 PRT Cellulasevariants 2 Gly Ser Gly His Thr Thr Arg Tyr Trp Asp Cys Cys Lys Pro SerCys 1 5 10 15 Ala Trp Asp Glu Lys Ala Ala Val Ser Arg Pro Val Thr ThrCys Asp 20 25 30 Arg Asn Asn Ser Pro Leu Ser Pro Gly Ala Val Ser Gly CysAsp Pro 35 40 45 Asn Gly Val Ala Phe Thr Cys Asn Asp Asn Gln Pro Trp AlaVal Asn 50 55 60 Asn Asn Val Ala Tyr Gly Phe Ala Ala Thr Ala Phe Pro GlyGly Asn 65 70 75 80 Glu Ala Ser Trp Cys Cys Ala Cys Tyr Ala Leu Gln PheThr Ser Gly 85 90 95 Pro Val Ala Gly Lys Thr Met Val Val Gln Ser Thr AsnThr Gly Gly 100 105 110 Asp Leu Ser Gly Thr His Phe Asp Ile Gln Met ProGly Gly Gly Leu 115 120 125 Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe GlyPhe Thr Phe Pro Gly 130 135 140 Asn Arg Tyr Gly Gly Thr Thr Ser Arg SerGln Cys Ala Glu Leu Pro 145 150 155 160 Ser Val Leu Arg Asp Gly Cys HisTrp Arg Tyr Asp Trp Phe Asn Asp 165 170 175 Ala Asp Asn Pro Asn Val AsnTrp Arg Arg Val Arg Cys Pro Ala Ala 180 185 190 Leu Thr Asn Arg Ser GlyCys Val Arg Ala 195 200 3 202 PRT cellulase variants 3 Gly Thr Gly ArgThr Thr Arg Tyr Trp Asp Cys Cys Lys Pro Ser Cys 1 5 10 15 Gly Trp AspGlu Lys Ala Ser Val Ser Gln Pro Val Lys Thr Cys Asp 20 25 30 Arg Asn AsnAsn Pro Leu Ala Ser Thr Ala Arg Ser Gly Cys Asp Ser 35 40 45 Asn Gly ValAla Tyr Thr Cys Asn Asp Asn Gln Pro Trp Ala Val Asn 50 55 60 Asp Asn LeuAla Tyr Gly Phe Ala Ala Thr Ala Phe Ser Gly Gly Ser 65 70 75 80 Glu AlaSer Trp Cys Cys Ala Cys Tyr Ala Leu Gln Phe Thr Ser Gly 85 90 95 Pro ValAla Gly Lys Thr Met Val Val Gln Ser Thr Asn Thr Gly Gly 100 105 110 AspLeu Ser Gly Asn His Phe Asp Ile Leu Met Pro Gly Gly Gly Leu 115 120 125Gly Ile Phe Asp Gly Cys Thr Pro Gln Trp Gly Val Ser Phe Pro Gly 130 135140 Asn Arg Tyr Gly Gly Thr Thr Ser Arg Ser Gln Cys Ser Gln Ile Pro 145150 155 160 Ser Ala Leu Gln Pro Gly Cys Asn Trp Arg Tyr Asp Trp Phe AsnAsp 165 170 175 Ala Asp Asn Pro Asp Val Ser Trp Arg Arg Val Gln Cys ProAla Ala 180 185 190 Leu Thr Asp Arg Thr Gly Cys Arg Arg Ala 195 200 4201 PRT Cellulase variants 4 Gly Ser Gly Lys Ser Thr Arg Tyr Trp Asp CysCys Lys Pro Ser Cys 1 5 10 15 Ala Trp Ser Gly Lys Ala Ser Val Asn ArgPro Val Leu Ala Cys Asp 20 25 30 Ala Asn Asn Asn Pro Leu Asn Asp Ala AsnVal Lys Ser Gly Cys Asp 35 40 45 Gly Gly Ser Ala Tyr Thr Cys Ala Asn AsnSer Pro Trp Ala Val Asn 50 55 60 Asp Asn Leu Ala Tyr Gly Phe Ala Ala ThrLys Leu Ser Gly Gly Thr 65 70 75 80 Glu Ser Ser Trp Cys Cys Ala Cys TyrAla Leu Thr Phe Thr Ser Gly 85 90 95 Pro Val Ser Gly Lys Thr Leu Val ValGln Ser Thr Ser Thr Gly Gly 100 105 110 Asp Leu Gly Ser Asn His Phe AspLeu Asn Met Pro Gly Gly Gly Val 115 120 125 Gly Leu Phe Asp Gly Cys LysArg Glu Phe Gly Gly Leu Pro Gly Ala 130 135 140 Gln Tyr Gly Gly Ile SerSer Arg Ser Glu Cys Asp Ser Phe Pro Ala 145 150 155 160 Ala Leu Lys ProGly Cys Gln Trp Arg Phe Asp Trp Phe Lys Asn Ala 165 170 175 Asp Asn ProGlu Phe Thr Phe Lys Gln Val Gln Cys Pro Ser Glu Leu 180 185 190 Thr SerArg Thr Gly Cys Lys Arg Ala 195 200 5 201 PRT Cellulase variants 5 GlySer Gly Gln Ser Thr Arg Tyr Trp Asp Cys Cys Lys Pro Ser Cys 1 5 10 15Ala Trp Pro Gly Lys Ala Ala Val Ser Gln Pro Val Tyr Ala Cys Asp 20 25 30Ala Asn Phe Gln Arg Leu Ser Asp Phe Asn Val Gln Ser Gly Cys Asn 35 40 45Gly Gly Ser Ala Tyr Ser Cys Ala Asp Gln Thr Pro Trp Ala Val Asn 50 55 60Asp Asn Leu Ala Tyr Gly Phe Ala Ala Thr Ser Ile Ala Gly Gly Ser 65 70 7580 Glu Ser Ser Trp Cys Cys Ala Cys Tyr Ala Leu Thr Phe Thr Ser Gly 85 9095 Pro Val Ala Gly Lys Thr Met Val Val Gln Ser Thr Ser Thr Gly Gly 100105 110 Asp Leu Gly Ser Asn Gln Phe Asp Ile Ala Met Pro Gly Gly Gly Val115 120 125 Gly Ile Phe Asn Gly Cys Ser Ser Gln Phe Gly Gly Leu Pro GlyAla 130 135 140 Gln Tyr Gly Gly Ile Ser Ser Arg Asp Gln Cys Asp Ser PhePro Ala 145 150 155 160 Pro Leu Lys Pro Gly Cys Gln Trp Arg Phe Asp TrpPhe Gln Asn Ala 165 170 175 Asp Asn Pro Thr Phe Thr Phe Gln Gln Val GlnCys Pro Ala Glu Ile 180 185 190 Val Ala Arg Ser Gly Cys Lys Arg Ala 195200 6 203 PRT Cellulase variants 6 Gly Ser Gly His Ser Thr Arg Tyr TrpAsp Cys Cys Lys Pro Ser Cys 1 5 10 15 Ser Trp Ser Gly Lys Ala Ala ValAsn Ala Pro Ala Leu Thr Cys Asp 20 25 30 Lys Asn Asp Asn Pro Ile Ser AsnThr Asn Ala Val Asn Gly Cys Glu 35 40 45 Gly Gly Gly Ser Ala Tyr Ala CysThr Asn Tyr Ser Pro Trp Ala Val 50 55 60 Asn Asp Glu Leu Ala Tyr Gly PheAla Ala Thr Lys Ile Ser Gly Gly 65 70 75 80 Ser Glu Ala Ser Trp Cys CysAla Cys Tyr Ala Leu Thr Phe Thr Thr 85 90 95 Gly Pro Val Lys Gly Lys LysMet Ile Val Gln Ser Thr Asn Thr Gly 100 105 110 Gly Asp Leu Gly Asp AsnHis Phe Asp Leu Met Met Pro Gly Gly Gly 115 120 125 Val Gly Ile Phe AspGly Cys Thr Ser Glu Phe Gly Lys Ala Leu Gly 130 135 140 Gly Ala Gln TyrGly Gly Ile Ser Ser Arg Ser Glu Cys Asp Ser Tyr 145 150 155 160 Pro GluLeu Leu Lys Asp Gly Cys His Trp Arg Phe Asp Trp Phe Glu 165 170 175 AsnAla Asp Asn Pro Asp Phe Thr Phe Glu Gln Val Gln Cys Pro Lys 180 185 190Ala Leu Leu Asp Ile Ser Gly Cys Lys Arg Ala 195 200 7 205 PRT Cellulasevariants 7 Gly Ile Gly Gln Thr Thr Arg Tyr Trp Asp Cys Cys Lys Pro SerCys 1 5 10 15 Ala Trp Pro Gly Lys Gly Pro Ser Ser Pro Val Gln Ala CysAsp Lys 20 25 30 Asn Asp Asn Pro Phe Asn Asp Gly Gly Ser Thr Arg Ser GlyCys Asp 35 40 45 Ala Gly Gly Ser Ala Tyr Met Cys Ser Ser Gln Ser Pro TrpAla Val 50 55 60 Ser Asp Glu Leu Ser Tyr Gly Trp Ala Ala Val Lys Leu AlaGly Ser 65 70 75 80 Ser Glu Ser Gln Trp Cys Cys Ala Cys Tyr Glu Leu ThrPhe Thr Ser 85 90 95 Gly Pro Val Ala Gly Lys Lys Met Ile Val Gln Ala ThrAsn Thr Gly 100 105 110 Gly Asp Leu Gly Asp Asn His Phe Asp Leu Ala IlePro Gly Gly Gly 115 120 125 Val Gly Ile Phe Asn Ala Cys Thr Asp Gln TyrGly Ala Pro Pro Asn 130 135 140 Gly Trp Gly Asp Arg Tyr Gly Gly Ile HisSer Lys Glu Glu Cys Glu 145 150 155 160 Ser Phe Pro Glu Ala Leu Lys ProGly Cys Asn Trp Arg Phe Asp Trp 165 170 175 Phe Gln Asn Ala Asp Asn ProSer Val Thr Phe Gln Glu Val Ala Cys 180 185 190 Pro Ser Glu Leu Thr SerLys Ser Gly Cys Ser Arg Ala 195 200 205 8 203 PRT Cellulase variants 8Thr Ala Gly Val Thr Thr Arg Tyr Trp Asp Cys Cys Lys Pro Ser Cys 1 5 1015 Gly Trp Ser Gly Lys Ala Ser Val Ser Ala Pro Val Arg Thr Cys Asp 20 2530 Arg Asn Gly Asn Thr Leu Gly Pro Asp Val Lys Ser Gly Cys Asp Ser 35 4045 Gly Gly Thr Ser Phe Thr Cys Ala Asn Asn Gly Pro Phe Ala Ile Asp 50 5560 Asn Asn Thr Ala Tyr Gly Phe Ala Ala Ala His Leu Ala Gly Ser Ser 65 7075 80 Glu Ala Ala Trp Cys Cys Gln Cys Tyr Glu Leu Thr Phe Thr Ser Gly 8590 95 Pro Val Val Gly Lys Lys Leu Thr Val Gln Val Thr Asn Thr Gly Gly100 105 110 Asp Leu Gly Asn Asn His Phe Asp Leu Met Ile Pro Gly Gly GlyVal 115 120 125 Gly Leu Phe Thr Gln Gly Cys Pro Ala Gln Phe Gly Ser TrpAsn Gly 130 135 140 Gly Ala Gln Tyr Gly Gly Val Ser Ser Arg Asp Gln CysSer Gln Leu 145 150 155 160 Pro Ala Ala Val Gln Ala Gly Cys Gln Phe ArgPhe Asp Trp Met Gly 165 170 175 Gly Ala Asp Asn Pro Asn Val Thr Phe ArgPro Val Thr Cys Pro Ala 180 185 190 Gln Leu Thr Asn Ile Ser Gly Cys ValArg Ala 195 200 9 203 PRT Cellulase variants 9 Thr Ser Gly Val Thr ThrArg Tyr Trp Asp Cys Cys Lys Pro Ser Cys 1 5 10 15 Ala Trp Thr Gly LysAla Ser Val Ser Lys Pro Val Gly Thr Cys Asp 20 25 30 Ile Asn Asp Asn AlaGln Thr Pro Ser Asp Leu Leu Lys Ser Ser Cys 35 40 45 Asp Gly Gly Ser AlaTyr Tyr Cys Ser Asn Gln Gly Pro Trp Ala Val 50 55 60 Asn Asp Ser Leu SerTyr Gly Phe Ala Ala Ala Lys Leu Ser Gly Lys 65 70 75 80 Gln Glu Thr AspTrp Cys Cys Gly Cys Tyr Lys Leu Thr Phe Thr Ser 85 90 95 Thr Ala Val SerGly Lys Gln Met Ile Val Gln Ile Thr Asn Thr Gly 100 105 110 Gly Asp LeuGly Asn Asn His Phe Asp Ile Ala Met Pro Gly Gly Gly 115 120 125 Val GlyIle Phe Asn Gly Cys Ser Lys Gln Trp Asn Gly Ile Asn Leu 130 135 140 GlyAsn Gln Tyr Gly Gly Phe Thr Asp Arg Ser Gln Cys Ala Thr Leu 145 150 155160 Pro Ser Lys Trp Gln Ala Ser Cys Asn Trp Arg Phe Asp Trp Phe Glu 165170 175 Asn Ala Asp Asn Pro Thr Val Asp Trp Glu Pro Val Thr Cys Pro Gln180 185 190 Glu Leu Val Ala Arg Thr Gly Cys Ser Arg Ala 195 200 10 235PRT Cellulase variants 10 Cys Asn Gly Tyr Ala Thr Arg Tyr Trp Asp CysCys Lys Pro His Cys 1 5 10 15 Gly Trp Ser Ala Asn Val Pro Ser Leu ValSer Pro Leu Gln Ser Cys 20 25 30 Ser Ala Asn Asn Thr Arg Leu Ser Asp ValSer Val Gly Ser Ser Cys 35 40 45 Asp Gly Gly Gly Gly Tyr Met Cys Trp AspLys Ile Pro Phe Ala Val 50 55 60 Ser Pro Thr Leu Ala Tyr Gly Tyr Ala AlaThr Ser Ser Gly Asp Val 65 70 75 80 Cys Gly Arg Cys Tyr Gln Leu Gln PheThr Gly Ser Ser Tyr Asn Ala 85 90 95 Pro Gly Asp Pro Gly Ser Ala Ala LeuAla Gly Lys Thr Met Ile Val 100 105 110 Gln Ala Thr Asn Ile Gly Tyr AspVal Ser Gly Gly Gln Phe Asp Ile 115 120 125 Leu Val Pro Gly Gly Gly ValGly Ala Phe Asn Ala Cys Ser Ala Gln 130 135 140 Trp Gly Val Ser Asn AlaGlu Leu Gly Ala Gln Tyr Gly Gly Phe Leu 145 150 155 160 Ala Ala Cys LysGln Gln Leu Gly Tyr Asn Ala Ser Leu Ser Gln Tyr 165 170 175 Lys Ser CysVal Leu Asn Arg Cys Asp Ser Val Phe Gly Ser Arg Gly 180 185 190 Leu ThrGln Leu Gln Gln Gly Cys Thr Trp Phe Ala Glu Trp Phe Glu 195 200 205 AlaAla Asp Asn Pro Ser Leu Lys Tyr Lys Glu Val Pro Cys Pro Ala 210 215 220Glu Leu Thr Thr Arg Ser Gly Met Asn Arg Ala 225 230 235 11 211 PRTCellulase variants 11 Gly Met Ala Thr Arg Tyr Trp Asp Cys Cys Leu AlaSer Ala Ser Trp 1 5 10 15 Glu Gly Lys Ala Pro Val Tyr Ala Pro Val AspAla Cys Lys Ala Asp 20 25 30 Gly Val Thr Leu Ile Asp Ser Lys Lys Asp ProSer Gly Gln Ser Gly 35 40 45 Cys Asn Gly Gly Asn Lys Phe Met Cys Ser CysMet Gln Pro Phe Asp 50 55 60 Asp Glu Thr Asp Pro Thr Leu Ala Phe Gly PheGly Ala Phe Thr Thr 65 70 75 80 Gly Gln Glu Ser Asp Thr Asp Cys Ala CysPhe Tyr Ala Glu Phe Glu 85 90 95 His Asp Ala Gln Gly Lys Ala Met Lys ArgAsn Lys Leu Ile Phe Gln 100 105 110 Val Thr Asn Val Gly Gly Asp Val GlnSer Gln Asn Phe Asp Phe Gln 115 120 125 Ile Pro Gly Gly Gly Leu Gly AlaPhe Pro Lys Gly Cys Pro Ala Gln 130 135 140 Trp Gly Val Glu Ala Ser LeuTrp Gly Asp Gln Tyr Gly Gly Val Lys 145 150 155 160 Ser Ala Thr Glu CysSer Lys Leu Pro Lys Pro Leu Gln Glu Gly Cys 165 170 175 Lys Trp Arg PheSer Glu Trp Gly Asp Asn Pro Val Leu Lys Gly Ser 180 185 190 Pro Lys ArgVal Lys Cys Pro Lys Ser Leu Ile Asp Arg Ser Gly Cys 195 200 205 Gln ArgAla 210 12 30 DNA Humicola grissea 12 gaatgacttg gttgacgcgt caccagtcac30 13 30 DNA Humicola grissea 13 gaatgacttg gttgagtact caccagtcac 30 1434 DNA Humicola grissea 14 cactggcggc gacctgggat ctaaccactt cgat 34 1537 DNA Humicola grissea 15 atcgaagtgg ttagatccca ggtcgccgcc tgtgctc 3716 20 DNA Humicola grissea 16 cgacttcaat gtccagtcgg 20 17 26 DNAHumicola grissea 17 gcgctctaga ggattaaagg cactgc 26 18 35 DNA Humicolagrissea 18 cgacctggga tcgaacgact tcgatatcgc catgc 35 19 30 DNA Humicolagrissea 19 cgactccagc ttccccgtct tcacgccccc 30 20 34 DNA Humicolagrissea 20 cgagcttcta gatctcgact agaggcactg ggag 34 21 27 DNA Humicolagrissea 21 ggatgccatg cttggaggat agcaacc 27 22 29 DNA Humicola grissea22 gggggcgtga agacgggaag ctggagtcg 29 23 1261 DNA Humicola grissea 23caagaacctc acactcattt tattcacgct catttattct aaaacttcaa tatgcgctct 60gctcctattt tccgcacggc cctggcggct gcgctccccc ttgccgcact cgccgccgat 120ggcaagtcga ccagatactg ggactgctgc aagccatcgt gctcttggcc cggaaaggca 180ctcgtgaacc agcctgtctt cacttgcgac gccaaattcc agcgcatcac cgaccccaat 240accaagtcgg gctgcgatgg cggctcggcc ttttcgtgtg ctgaccagac cccctgggct 300ctgaacgacg atgtcgccta tggcttcgct gccacggcta tttcgggtgg atcggaagcc 360tcgtggtgct gcgcatgcta cgctcttact ttcacctcgg gccctgtggc cggcaagacc 420atggtcgtcc agtcgaccaa caccggcggc gatctcggca gcaaccattt cgacctccag 480attccaggcg gcggtgtcgg catctttgat gggtgcaccc cccagttcgg aggtctcgct 540ggcgaacgct acggtggcat ctcagaccgc agctcctgcg actcgttccc tgcggcgctc 600aagcccggct gcctctggcg cttcgattgg ttcaagaacg ccgacaaccc gacctttacc 660ttcaagcagg tgcagtgccc cgccgagctt gttgccagga ccggctgcaa gcgcgaggat 720gacggcaact tccccgtctt cacgcccccc gcgggtagca acaccggcgg tagccagtcg 780agctccacta tcgcttccag ctcgacctcc aaggctcaga cttcggccgc cagctccacc 840tccaaggctg tcgtgactcc cgtctccagc tccacctcga aggccgctga ggtccccaaa 900tccagctcga cctccaaggc tgccgaggtc gccaagccca gctcaacttc gacctcgacc 960tcgacctcga ccaaggtcag ctgctctgcg accggtggct cctgcgtcgc tcagaagtgg 1020gcgcagtgcg gcggcaatgg cttcaccggc tgcacgtcgt gcgtcagcgg caccacctgc 1080cagaagcaaa atgactggta ctcccagtgc ctctaagtcg tttgtagtag cagtttgaag 1140gatgtcaggg atgagggagg gaggagtggg ggaaaagtac gccgcagttt tttggtagac 1200ttactgtatt gttgagtaat tacccattcg cttcttgtac gaaaaaaaaa aaaaaaaaaa 1260 a1261 24 41 DNA Humicola grissea 24 gctgcaagcc gtcctgtggc tggagcgctaacgtgcccgc g 41 25 36 DNA Humicola grissea 25 cgatgtttcc ggaggccactttgacattct ggttcc 36 26 30 DNA Humicola grissea 26 gaatgacttg gttgacgcgtcaccagtcac 30

What is claimed is:
 1. An enzyme variant comprising a catalytic coredomain exhibiting cellulolytic activity, which variant is derived from anaturally occurring parental cellulase by amino acid residuesubstitution, insertion or deletion or any combination thereof, and atposition 5 (cellulase numbering) holds an alanine residue (A), a serineresidue (S), or a threonine residue (T); at position 8 (cellulasenumbering) holds a phenylalanine residue (F), or a tyrosine residue (Y);at position 9 (cellulase numbering) holds a phenylalanine residue (F), atryptophan residue (W), or a tyrosine residue (Y); at position 10(cellulase numbering) holds an aspartic acid residue (D); and atposition 121 (cellulase numbering) holds an aspartic acid residue (D).2. The variant according to claim 1, which at position 119 (cellulasenumbering) holds an amino acid residue selected from the groupconsisting of histidine (H), aspartic acid (D), asparagine (N),glutamine (Q), arginine (R), and phenylalanine (F).
 3. The variantaccording to claim 1, which at position 6 (cellulase numbering) holds anamino acid residue selected from the group consisting of threonine (T)and serine (S).
 4. The variant according to claim 1, which at position 7(cellulase numbering) holds an amino acid residue selected from thegroup consisting of arginine (R), leucine (L), isoleucine (I),tryptophan (W), and lysine (K).
 5. A cellulase variant, which variantholds 4 or more of the following disulfide bridges: C11-C135; C12-C47;C16-C86; C31-C56; C87-C199; C89-C189; and C156-C167 (cellulasenumbering).
 6. The cellulase variant according to claim 5, which variantholds 5 or more of the following disulfide bridges: C11-C135; C12-C47;C16-C86; C31-C56; C87-Cl99; C89-C189; and C156-C167 (cellulasenumbering).
 7. The cellulase variant according to claim 5, which variantholds 6 or more of the following disulfide bridges: C11-C135; C12-C47;C16-C86; C31-C56; C87-Cl99; C89-C189; and C156-C167 (cellulasenumbering).
 8. The cellulase variant according to claim 5, in whichvariant a cysteine residue has been replaced by a different naturalamino acid residue at one or more of the positions 16, 86, 87, 89, 189,and/or 199 (cellulase numbering).
 9. The cellulase variant according toclaim 5 selected from the group consisting the following variantsderived from Humicola insolens endoglucanase V (EGV): C12G/C47M, C47G,C87M/C199G and C16M/C86G.
 10. A method of reducing the thermostabilityof a cellulase comprising removal, by amino acid substitution, deletionor insertion, of one or more disulfide bridges selected from the groupconsisting of C11-C135; C12-C47; C16-C86; C31-C56; C87-C199; C89-C189;and C156-C167 (cellulase numbering).
 11. A cellulase variant derivedfrom a parental cellulase by substitution, insertion and/or deletion atone or more amino acid residues located in the substrate binding cleftat a position within an enzyme-substrate interactive distance from thesubstrate.
 12. The cellulase variant according to claim 11, whichvariant has been derived from a parental cellulases by substitution,insertion and/or deletion at one or more amino acid residues located inthe substrate binding cleft at a distance of not more than 5 Å from thesubstrate.
 13. The cellulase variant according to claim 12, whichvariant has been derived from a parental cellulase by substitution,insertion and/or deletion at one or more of the following positions: 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 21a, 42, 44,45, 47, 48, 49, 49a, 49b, 74, 82, 95j, 110, 111, 112, 113, 114, 115,116, 119, 121, 123, 127, 128, 129, 130, 131, 132, 132a, 133, 145, 146,147, 148, 149, 150b, 178, and/or 179 (cellulase numbering).
 14. Thecellulase variant according to claim 12, which variant has been derivedfrom a parental cellulase by substitution, insertion and/or deletion atone or more of the following positions: 4, 5, 13, 14, 15, 16, 19, 20,21, 21a, 42, 44, 47, 48, 49, 49a, 49b, 74, 82, 95j, 110, 111, 113, 115,116, 119, 123, 129, 131, 132a, 133, 145, 146, 150b, 178, and/or 179(cellulase numbering).
 15. The cellulase variant according to claim 13selected from the group consisting the following variants derived fromHumicola insolens endoglucanase V (EGV): T6S, R7I, R7W, Y8F, W9F,C12M/C47G, W18Y, W18F, S45T, S45N, D114N, F132D, Y147D, Y147C, Y147W,Y147V, Y147R, Y147G, Y147Q, Y147N, Y147K, Y147H, Y147F and Y147S. 16.The cellulase variant according to claim 13 selected from the groupconsisting the following variants derived from Humicola insolensendoglucanase V (EGV): R4H, R4Q, K13L, K13R, K13Q, P14A, P14T, S15T,S15H, C16M/C86G, A19P, A19T, A19G, A19S, K20G, D42Y, D42W, C47G, E48D,E48Q, E48D/P49*, E48N/P49*, S110N, L115I, G116D, H119R, H119Q, H119F,N123Å, N123M, N123Q, N123Y, N123D, V129L, D133N and D178N.
 17. Thecellulase variant according to claim 11, which variant has been derivedfrom a parental cellulase by substitution, insertion and/or deletion atone or more amino acid residues located in the substrate binding cleftat a distance of not more than 3 Å from the substrate.
 18. The cellulasevariant according to claim 17, which variant has been derived from aparental cellulase by substitution, insertion and/or deletion at one ormore of the following positions: 6, 7, 8, 10, 12, 13, 14, 15, 18, 20,21, 45, 48, 74, 110, 111, 112, 113, 114, 115, 119, 121, 127, 128, 129,130, 131, 132, 132a, 146, 147, 148, 150b, 178, and/or 179 (cellulasenumbering).
 19. The cellulase variant according to claim 17, whichvariant has been derived from a parental cellulase by substitution,insertion and/or deletion at one or more of the following positions: 13,14, 15, 20, 21, 48, 74, 110, 111, 113, 115, 119, 129, 131, 146, 150b,178, and/or 179 (cellulase numbering).
 20. A cellulase variant, in whichvariant an amino acid residue has been changed into a conserved aminoacid residue at one or more positions according to Table 1, at whichposition(s) between 7 and 10 amino acid residues of the 11 residuesidentified in Table 1, are identical.
 21. The cellulase variantaccording to claim 20, which variant has been derived from a parentalcellulases by substitution, insertion and/or deletion at one or more ofthe following positions: 13, 14, 15, 20, 21, 22, 24, 28, 32, 34, 45, 48,50, 53, 54, 62, 63, 64, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 79, 85,88, 90, 92, 93, 95, 96, 97, 98, 99, 104, 106, 110, 111, 113, 115, 116,118, 119, 131, 134, 138, 140, 146, 152, 153, 163, 166, 169, 170, 171,172, 173, 174, 174, 177, 178, 179, 180, 193, 196, and/or 197 (cellulasenumbering).
 22. A cellulase variant according to claim 21 comprising oneor more of the following mutations (cellulase numbering): K13L or L13K;P14Å or A14P; S15H or H15S; K20E, K20G, K20A, E20K, G20K, A20K, E20G,E20A, G20E, A20E, G20A, or A20G; K21N or N21K; A22G, A22P, G22A, P22A,G22P, or P22G; V24*, V24L, *24V, L24V, *24L, or L24*; V28A, V28L, A28V,L28V, A28L, or L28A; N32D, N32S, N32K, D32N, S32N, K32N, D32S, D32K,S32D, K32D, S32K, or K32S; N34D or D34N; I38L, I38F, I38Q, L38I, F38I,Q38I, L38F, L38Q, F38L, Q38L, F38Q, or Q38F; S45N or N45S; G46S or S46G;E48D, E48N, D48E, N48E, D48N, or N48D; G50N or N50G; A53S, A53G, A53K,S53A, G53A, K53A, S53G, S53K, G53S, K53S, G53K, or K53G; Y54F or F54Y;W62F or F62W; A63D or D63A; V64I, V64D, I64V, D64V, I64D, or D64I; N65S,N65D, N65E, S65N, D65N, E65N, S65D, S65E, D65S, E65S, D65E, or E65D;D66N, D66P, D66T, N66D, P66D, T66D, N66P, N66T, P66N, T66N, P66T, orT66P; F68V, F68L, F68T, F68P, V68F, L68F, T68F, P68F, V68L, V68T, V68P,L68V, T68V, P68V, L68T, L68P, T68L, P68L, T68P, or P68T; A69S, A69T,S69A, T69A, S69T, or T69S; L70Y or Y70L; G71A or A71G; F72W, F72Y, W72F,Y72F, W72Y, or Y72W; A73G or G73A; A74F or F74A; T75V, T75A, T75G, V75T,A75T, G75T, V75A, V75G, A75V, G75V, A75G, or G75A; G79T or T79G; W85T orT85W; A88Q, A88G, A88R, Q88A, G88A, R88A, Q88G, Q88R, G88Q, R88Q, G88R,or R88G; Y90For F90 Y; L92A or A92L; T93Q, T93E, Q93T, E93T, Q93E, orE93Q; T95E or E95T; S96T or T96S; G97T, G97A, T97G, A97G, T97A, or A97T;P98A or A98P; V99L or L99V; M104L or L104M; V106F or F106V; S110N orN110S; T111I, T111V, I111T, V111T, I111V, or V111I; G113Y or Y113G;L115V or V115L; G116S, G116Q, S116G, Q116G, S116Q, or Q116S; N118T,N118G, N118Q, T118N, G118N, Q118N, T118G, T118Q, G118T, Q118T, G118Q, orQ118G; H119Q, H119N, Q119H, or N119H; V129L or L129V; I131L, I131A,L131I, A131I, L13:LA, or A131L; G134A or A134G; Q138E or E138Q; G140N orN140G; R146Q or Q146R; S152D or D152S; R153K, R153L, R153A, K153R,L153R, A153R, K153L, K153A, L153K, A153K, L153A, or A153L; L163V, L163W,V163L, W163L, V163W, or W163V; G166S or S166G; W169F or F169W; R170F orF170R; F171Y, F171A, Y171F, A171F, Y171A, or A171Y; D172E, D172S, E172D,S172D, E172S, or S172E; W173E or E173W; F174M, F174W, M174F, W174F,M174W, or W174M; A177N or N177A; D178P or P178D; N179V or V179N; P180Lor L180P; L193I or I193L; R196I, R196K, I196R, K196R, I196K, or K196I;and/or T197S or S197T.
 23. A cellulase variant, which variant has beenderived from a parental cellulase by substitution, insertion and/ordeletion at one or more of the following positions (cellulasenumbering), and which variant: in position 4 holds R, H, K, Q, V, Y, orM; in position 5 holds S, T, or A; in position 13 holds K, or L; inposition 14 holds P, or A; in position 15 holds H, or S; in position 16holds C, or A; in position 19 holds A, D, S, P, T, or E; in position 20holds A, E, G, or K; in position 21 holds K, or N; in position 21a holdsV or *; in position 22 holds A, G, or P; in position 24 holds L, V, or*; in position 28 holds A, L, or V; in position 32 holds D, K, N, or S;in position 34 holds D or N; in position 38 holds F, I, L, or Q; inposition 42 holds D, G, T, N, S, K, or *; in position 44 holds K, V, R,Q, G, or P; in position 45 holds N, or S; in position 46 holds G, or S;in position 47 holds C, or Q; in position 48 holds D, E, N, or S; inposition 49 holds P, S, A, G, or *; in position 49a holds C, or *; inposition 49b holds N, or *; in position 50 holds G, or N; in position 53holds A, G, K, or S; in position 54 holds F, or Y; in position 62 holdsF, or W; in position 63 holds A, or D; in position 64 holds D, I, or V;in position 65 holds D, I, N, or S; in position 68 holds D, N, P, or T;in position 69 holds A, S, or T; in position 70 holds L, or Y; inposition 71 holds A, or G; in position 72 holds F, W, or Y; in position73 holds A, or G; in position 74 holds A, or F; in position 75 holds A,G, T, or V; in position 79 holds G, or T; in position 82 holds F, or *;in position 88 holds A, G, Q, or R; in position 90 holds F, or Y; inposition 92 holds A, or L; in position 93 holds E, Q, or T; in position95 holds E, or *; in position 95j holds P, or *; in position 96 holds S,or T; in position 97 holds A, G, or T; in position 98 holds A, or P; inposition 99 holds L, or V; in position 104 holds L, or M; in position106 holds F, or V; in position 110 holds N, or S; in position 111 holdsI, T, or V; in position 113 holds G, or Y; in position 115 holds L, orV; in position 116 holds G, Q, or S; in position 118 holds G, N, Q, orT; in position 119 holds H, N, or Q; in position 129 holds L, or V; inposition 131 holds A, I, or L; in position 132 holds A, P, or T; inposition 133 holds D, K, N, or Q; in position 134 holds A, or G; inposition 138 holds E, or Q; in position 145 holds A, D, N, or Q; inposition 146 holds Q, or R; in position 150b holds A, or *; in position152 holds D, or S; in position 153 holds A, K, L, or R; in position 163holds L, V, or W; in position 166 holds G, or S; in position 169 holdsF, or W; in position 170 holds F, or R; in position 171 holds A, F, orY; in position 172 holds D, E, or S; in position 173 holds E, or W; inposition 174 holds F, V, or W; in position 177 holds A, or N; inposition 178 holds D, or P; in position 179 holds N, or V; in position180 holds L, or P; in position 193 holds I, or L; in position 196 holdsI, K, or R; and/or in position 197 holds S, or T.
 24. A cellulasevariant having an altered anion tenside sensitivity, and which variantis from a parental cellulase by substitution, insertion and/or deletionat one or more of the following positions: 2, 4, 7, 8, 10, 13, 15, 19,20, 21, 25, 26, 29, 32, 33, 34, 35, 37, 40, 42, 42a, 43, 44, 48, 53, 54,55, 58, 59, 63, 64, 65, 66, 67, 70, 72, 76, 79, 80, 82, 84, 86, 88, 90,91, 93, 95, 95d, 95h, 95j, 97, 100, 101, 102, 103, 113, 114, 117, 119,121, 133, 136, 137, 138, 139, 140a, 141, 143a, 145, 146, 147, 150e,150j, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160c, 160e, 160k,161, 162, 164, 165, 168, 170, 171, 172, 173, 175, 176, 178, 181, 183,184, 185, 186, 188, 191, 192, 195, 196, 200, and/or 201 (cellulasenumbering).
 25. A cellulase variant, in which variant an amino acidresidue has been substituted at one or more of the following positions:17, 85, 86 87, 88, and/or 89 (cellulase numbering).
 26. A Humicolainsolens EGV variant, in which one or more of the following mutationshave been introduced: D42W, D42Y, or L70Y.
 27. A Thielavia terrestriscellulase variant, in which variant one or more of the followingmutations have been introduced: P19A, G20K, Q44K, N48E, Q119H or Q146 R.28. The Thielavia terrestris/Q119H variant.
 29. A Pseudomonasfluorescens cellulase variant, in which variant one or more of thefollowing mutations have been introduced: Y4R, H15S, N119Q or Q146R. 30.A Crinipellis scabella cellulase variant, in which one or more of thefollowing mutations have been introduced: V4R, T132a*, Q133D or Q146R.31. A method for improving the properties of a cellulolytic enzyme byamino acid substitution, deletion or insertion, the method comprisingthe steps of: a. constructing a multiple alignment of at least two aminoacid sequences known to have three-dimensional structures similar toendoglucanase V (EGV) from Humicola insolens known from Protein DataBank entry 4ENG; b. constructing a homology-built three-dimensionalstructure of the cellulolytic enzyme based on the structure of the EGV;c. identifying amino acid residue positions present in a distance fromthe substrate binding cleft of not more than 5 Å; d. identifyingsurface-exposed amino acid residues of the enzyme; e. identifying allcharged or potentially charged amino acid residue positions of theenzyme; f. choosing one or more positions wherein the amino acid residueis to be substituted, deleted or where an insertion is to be provided;and g. carrying out the substitution, deletion or insertion by usingconventional protein engineering techniques.
 32. The method according toclaim 31, wherein step f. is carried out by choosing positions which, asa result of the alignment of step a., carry the same amino acid residuein a majority of the aligned sequences.
 33. The method according toclaim 32, wherein step f is carried out by choosing positions which, inthe aligned sequences, carries different amino acid residues.
 34. Amethod of improving the specific activity of a naturally occurringparental cellulase by carrying out a substitution, deletion or insertionat amino acid residue positions present in a distance from the substratebinding cleft of not more than 5 Å.
 35. A method of altering the pHactivity profile, the pH activity optimum, the pH stability profile, orthe pH stability optimum of a naturally occurring parental cellulase byaltering the electrostatic environment either locally or globally bycarrying out a substitution, deletion or insertion at amino acid residuepositions present either in a distance from the substrate binding cleftof not more than 5 Å, or at surface-exposed amino acid residue positionsof the enzyme; preferably by a substitution involving a charged orpotentially charged residue, this residue either being the originalresidue or the replacement residue.
 36. A method of altering thestability of a naturally occurring parental cellulase in the presence ofan anionic tenside or anionic detergent component by altering theelectrostatic environment either locally or globally by carrying out asubstitution, deletion or insertion at one or more surface-exposed aminoacid residue positions of the enzyme.